Mettler Toledo PureSpeed Operating instructions

Brand: Mettler Toledo

Model: PureSpeed

Type: Operating instructions

Guide to PureSpeed

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Protein Tips

A Quick Reference Guide

Reference – PureSpeed

PureSpeed Quick Reference Topics

PureSpeed Performance: The Tip Concentrating Effect ....................................................... 3

PureSpeed Tips ............................................................................................................ 4

PureSpeed Resin .......................................................................................................... 6

PureSpeed Hardware .................................................................................................... 7

Afnity Capture ProA, ProG and IMAC Resins ................................................................... 8

Getting Started: Sample and Tip Prep and Purication Method Design .............................. 10

Protein Recovery ....................................................................................................... 10

Protein Purity ............................................................................................................. 12

Protein Activity ........................................................................................................... 13

Optimization and Enhancement of the Purication Process ............................................. 14

Purespeed, ColorTrak Guide and Rainin are trademarks of Rainin Instrument, LLC.

Tip Concentrating Effect, Capture Purify Enrich, PhyTip and PhyNexus are trademarks of PhyNexus, Inc.

PureSpeed

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Performance: The Tip Concentrating Effect

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What is the Tip Concentrating Effect of PureSpeed tips?

The Tip Concentrating Effect is a process that allows the recovery of high concentrations of proteins in a single purication

procedure

. PureSpeed pipette tips are designed to produce very high concentrations of recovered proteins compared to

competitive techniques such as spin columns, gravity columns, magnetic beads and FPLC.

The Tip Concentrating Effect relies on the ability of PureSpeed tips to capture protein on very small resin bed volumes. The

proteins are then recovered in a highly efcient and effective process. All conditions being equal, with any two different

resin bed sizes of PureSpeed tips, the smaller resin bed size will produce the highest concentration of recovered protein.

How does the Tip Concentrating Effect work? It seems counter intuitive to increase the concentration of a protein by

using a smaller tip. How is this possible?

Yes, it is counter intuitive. It is also no surprise that with all other technologies the recovered protein becomes more dilute

as the resin bed size is decreased (competitive technologies are discussed on pages 4 and 8). PureSpeed tips are

unique: they are designed to use the same elution-volume-to-bed ratio as the bed volume is decreased, usually a

3x ratio. Even with resin bed sizes as low as 5 µL (a 100-fold decrease over a “normal” 0.5 mL resin bed) a 3x (or

even a 2x) elution volume can be used. Thus, an 80 µL bed uses a 240 µL elution volume; a 20 µL bed uses a 60 µL

elution volume; a 5 µL bed uses a 15 µL elution volume, and so on. For example, to increase the concentration of a

recovered sample, all capture and wash conditions are kept the same but the resin bed volume of the tip is decreased

from 80 µL to 20 µL. Essentially, the same amount of protein is captured with 20 µL bed vs. the original 80 µL bed, but

now is eluted with 60 µL of buffer rather than the original 240 µL. Thus, the concentration of the recovered protein can be

increased up to 4 fold over the original, larger bed size purication process.

What is meant by optimizing and enhancing the capture step and elution step? Is this part of the Tip Concentrating

Effect process?

Yes, it is important, even necessary, that the PureSpeed tips operate at the highest possible performance. First, PureSpeed

tips operate under a back and forth ow process, continuously owing the sample through the resin bed. Target proteins

that are present in the sample are transported actively to the afnity resin bed with sufcient cycles to increase contact

time to fully capture the protein. This active transport process increases the capture kinetics (over diffusion processes)

and also allows the capture equilibrium reaction to reach completion.

By optimizing and enhancing the capture process, even small resin beds can be used to capture the protein contained in

the sample. Resin beds are loaded to a very high percentage of available capture sites and substantial amounts of pro-

tein can be captured with small bed volumes. Finally, after washing to remove contaminants from the resin bed, a very

efcient, small volume elution process is used to enrich the sample. Since only very small elution volumes are needed to

elute the proteins from the resin bed, the recovered protein concentration is very high. As the elution volume is decreased,

the concentration of protein is increased. PureSpeed tips can be eluted with extremely small volumes of buffer. The con-

centrations of recovered proteins are 5-20 times higher than what can be produced from competitive technologies.

D. T. Gjerde, Concentrating Effect of Pipette Tip Columns, submitted (2012).

If smaller bed volume PureSpeed tips give higher concentration, why not always use the smallest bed tip available?

Because many times, the total mass of recovered protein is important too. In samples where the initial protein concentra-

tions are high, higher bed volumes should be used to prevent overloading of the tip. The back and forth loading method

allows the resin to be loaded with protein to very high levels. However if too much protein is present, some protein can be

lost in the tip ow through. Overloading can be monitored by performing an analysis of the capture ow-through before

and after capture.

Why do spin columns, gravity columns and FPLC use large bed volumes?

Competitive technologies have less efcient capture processes. The control of uid through the column in the capture step

is less efcient. Spin columns need a larger resin bed to give a better chance of capture. The ow through a spin column

is extremely rapid giving limited time for protein capture in only a single chance – a single uid pass. Proteins need suf-

cient time to interact with the resin to be captured. Increasing the bed size of a spin column bed increases the opportunity

for capture in an inefcient process. Gravity columns and FPLC have less control over the uid control than PureSpeed

pipette and tip uid control. With competitive techniques it is impossible to use very small capture volumes. The same is

true for the elution step in competitive products. Inefcient elution processes require large elution volume to be effective.

These large buffer volumes dilute the recovered protein. PureSpeed tips use very small elution volumes, which incidentally

is why the elution process for PureSpeed tips is sometimes called the enrichment process.

Why do PureSpeed tips outperform magnetic beads?

For the same amount of magnetic bead resin, smaller elution volumes can be used with PureSpeed tips making the

recovered protein much higher in concentration. But there are other reasons PureSpeed tips outperform magnetic beads.

Magnetic beads are low capacity – only the surface is derivatized so capacity is lower. Also, magnetic beads cannot use

back and forth ow, the active transport mechanism of PureSpeed tips, so the capture process is slower. The washing/

purication process for PureSpeed tips is more effective; therefore, the recovered protein is purer.

PureSpeed Tips

What are PureSpeed Protein Tips?

PureSpeed Protein Tips are part of a unique protein purication and enrichment system offered by Rainin. The complete

system includes the E4 XLS Rainin pipette, a base, ColorTrak Guide and PureSpeed Protein Tips to automate the process

of Capture, Purify and Enrich for proteins. The PureSpeed tips are novel tip protein capture devices employing various

afnity resins maintained at the base outlet of a pipette tip. PureSpeed tips capture the protein of interest by bidirectional

ow of sample volume through the tip bed. By using back and forth ow, the capture interaction of the protein with the tip

is allowed to reach completion: capture is complete.

Why would I use PureSpeed Protein Tips?

PureSpeed Protein Tips have been optimized to produce the highest possible purity, yield, concentration and activity of

sample proteins. They have also been optimized to be able to perform these functions with low volume samples, thus

saving valuable resources. Sample work ow is seamlessly integrated with protein expression and assays, with complete

sample and solution tracking. The tips can operate rapidly in parallel operation to perform purication in minimal time,

frequently in as little as 15 minutes. The tips are easy to set up and are disposable after use making buffer preparation

and cleanup very easy.

How long can refrigerated ProA, ProG and IMAC PureSpeed Protein Tips be stored?

Each PureSpeed tip is manufactured and shipped with a special bead coating liquid to maintain bead hydration and

prevent contamination. The tips are guaranteed to remain stable for at least six months after delivery, though will remain

stable, hydrated and usable for many more months.

If I let the ProA PureSpeed Protein Tip dry out while I’m using it, will the captured protein be affected?

For optimal recovery and to avoid protein denaturation, PureSpeed Protein Tips should remain hydrated through the entire

purication process. The tips should be stored as shipped with the coating liquid to prevent the resin bed from drying.

Once the purication process has been initiated, the tips will not dry out if used under recommended operating condi-

tions.

Why ship 12 PureSpeed Protein Tips in a small container?

PureSpeed Protein Tips are packed as a group of 12 tips in order to maintain the industry standard of 9 mm center-to-

center spacing. This spacing is used in many products include 96 well plates and sample stands. Depending on the E4

XLS pipette used, up to 8 or 12 tips can be used in parallel. Versatile, parallel operation can be best appreciated as the

researcher designs research experiments for optimum work ow. For those researchers who only require 1 or 2 samples

to be puried per run, this packaging offers a convenient size for longer term storage of the product.

These tips look easy to make. Can I just make them in my lab?

Looks can be deceiving, but we take that question as a compliment. Why? Because we have found that the highest

level of sophistication can be found in the simplest looking, simplest to use and highest performing devices. Rather than

explaining this further, we will simply direct the reader to just one example: the

Tip Concentrating Effect

of the PureSpeed

tips (described on page 3.) The features and method of use of the technology are the subject of several issued patents

and patents pending.

How are PureSpeed Protein Tips QC tested?

PureSpeed Protein Tips are tested at multiple steps in manufacture for backpressure, ow rate and dimensions. The resins

in the tip have been tested for capacity.

Can I reuse my PureSpeed Protein Tips?

PureSpeed tips are priced to be disposable and we do not recommend that they be reused. We cannot guarantee that

contaminating proteins will not be eluted in the new experiment. The capture efciency or the recovery of enriched protein

from a used tip cannot be guaranteed. After use, tips will dry out, harming tip capacity and potentially contaminating the

resin with bacteria.

What is the resin volume in each of the PureSpeed Protein Tips?

The resin bed is 20 or 80 µL for 1000 PureSpeed Protein Tips and 5 or 20 µL for 200 PureSpeed Protein Tips.

PureSpeed Resins

Can I buy PureSpeed Protein Tips with other resins?

Rainin currently supplies PureSpeed Protein Tips to capture proteins including the following resins: Protein A, Protein G

and IMAC. New resins are being considered. Please contact Rainin customer support for plans to add resins to the prod-

uct offering.

Can I purify my GST tagged proteins?

At present we do not carry a PureSpeed Protein Tip that has the appropriate resin to capture a GST tagged protein. We

are very interested in understanding your need a little further and are considering adding this afnity resin to our product

portfolio. For more information please contact your Rainin representative or Rainin customer support.

How do I know if I am using the right afnity resin? For example, I am using a mammalian expression system and

isolating mouse IgG3? What resin should I use?

Choosing the correct resin for a particular application may seem like a simple matter. It usually is, but sometimes a little

extra care may be needed. In the example posed in the question, ProA may not be the optimal resin for isolating mouse

IgG3. We recommend using ProG afnity resin, which has a stronger afnity for this particular protein over ProA resin. In

addition, mammalian expression systems frequently do not produce very large quantities of the desired protein, so chang-

ing to a resin with a higher afnity may allow you to capture more of the product.

How do I know if my resin is capturing protein? How do I know if my resin is saturated with protein?

Used correctly, PureSpeed tips can be loaded with a surprising amount of protein. Nevertheless, in cases where the resin

has the incorrect selectivity no protein may be captured. In other cases, protein is captured well loading up the resin, but

not all of the protein is collected from the sample volume. This is difcult to observe directly as most proteins are colorless

and only after the fact can capture information be determined. Because of parallel operation of PureSpeed tips, the user

is in complete control. Protein control standards can be run in parallel with the sample to ensure correct operation and

purication chemistry. In a broader experiment researchers will retain some of the original material, the “ow-through”

from each step (this is the material left after a step) so that a gel can be run at the end of the experiment to observe not

only the end product – the puried, concentrated protein, but also if any of the protein of interest came through in the ow

through. Reviewing this information allows the researcher to determine the success of the capture, wash and ultimately

the elution steps.

What is the IMAC resin made of and why does my IMAC resin have a blue color?

PureSpeed Protein Tips with IMAC resin are made of Ni-NTA (chelating) functional group bonded to an agarose resin

substrate. The nickel cation, held by the NTA (nitrilotriacetate) chelating group, has an afnity for 6-HIS tag portion of a

recombinant protein. There are two basic IMAC resins available: a nickel based and a cobalt based system. Cobalt resins

have a slight pink/red tinge to the resin, but the nickel resin with a bluish color tinge is the most common type of IMAC

resin.

PureSpeed Hardware

Will the PureSpeed Protein Tips t onto any pipettor we have in the lab?

No. PureSpeed Protein Tips are designed for use only with the LTS version of the E4 XLS electronic pipette. Why do we

match the PureSpeed tip to the E4 XLS pipette? We use the electronic capability of the pipette to simplify operation and

enhance performance of the tip. Rainin’s E4 XLS pipette has a dedicated PureSpeed mode that enables protein purication

to be programmed and controlled in a semi-automatic process.

Can PureSpeed Protein Tips be used on full automation robotics?

For full automation robotics, tips are available through PhyNexus (www.phynexus.com.) PhyNexus sells/supports a

complete line of tips and chemistry for automated robotics including platforms from PhyNexus, Tecan, Caliper, Dynamic

Devices, Beckman, Hamilton, Perkin Elmer and Agilent.

How do I program my pipette? For example, can I elute with an elution volume that is less than 3x of the resin tip

bed volume – is it hard to program?

Various default method protocols are built into the pipette software and excellent results can be obtained with no method

modications. However, it is possible to make small programming adjustments or even major adjustments to make a

new method, then save the method for immediate and/or future use. For example, it is very easy to program the pipette to

elute with only 2x the resin volume (rather than the default 3x volume) in order to produce the highest concentration pos-

sible of recovered protein. In another example, two elution volumes of 2x size may be used for elution. This will recover

protein in a very high concentration in the rst aliquot and elute the rest of the protein in the next aliquot. The pipette is

easily programmed for any elution volume and any number of elution aliquots.

Why is ow rate important when operating the PureSpeed Protein Tips?

A critical component of the capture step requires that the target sample protein has sufcient time to bind to the afnity

resin. By using a recommended slower ow rate, maximum capture of the desired protein can occur because this in-

creases residence time of the sample protein with the resin matrix. The default protocol utilizes a residence time of 6 and

18 minutes for 200 µL and 1000 µL samples, respectively. The capture efciency can also be increased by increasing

the number of capture cycles.

What materials are used to make a PureSpeed tip’s body and frit?

PureSpeed tip bodies are manufactured from polypropylene and the frits are mesh/screen retainers composed of a hydro-

philic, biocompatible polymer. The tip hardware is designed to have minimal contact with the sample protein to reduce

protein denaturation. This is accomplished by having protein-compatible surfaces and by reducing the overall exposed

surface area of the tip. The frits have an extremely low exposed surface area.

Are PureSpeed Protein Tips sterile?

No. PureSpeed Protein Tips are shipped in glycerol which is a bactericide. Prior to use, the tips can be stored at room

temperature for several hours with no degradation to the tip.

What are the current competitive technologies used for afnity purication?

Current afnity purication tools include spin columns, gravity columns, magnetic beads and the FPLC chromatography

platform. For small samples, PureSpeed tips outperform all of these technologies with higher protein concentrations,

higher purity and higher protein activity. The FPLC chromatography platforms perform well, but the throughput is limited

(samples are run one at a time). FPLC set-up and clean-up is time consuming and elution volumes can be large, diluting

the nal product.

Afnity Capture ProA, ProG and IMAC Resins

What is afnity purication?

Afnity resins are designed to be selective to a particular protein tag or protein property or characteristic. Recombinant pro-

teins are normally puried using a tag on the protein and an afnity resin that is selective to the particular tag. His-tagged

proteins are puried on IMAC afnity resins. Antibodies are puried with Protein A or Protein G afnity resins. There are

many other types of afnity resins including other tag selective resins or ion exchange resins. Proteins in a particular pH

environment have a specic charge and can be puried with ion exchange afnity resins. The combination of afnity and

specicity has been exploited to generate straightforward afnity purication methods.

What factors are important in PureSpeed sample capture?

Of course the afnity chemistry must be suitable to selectively capture the protein of interest. For example, the Protein A

resin will capture Mouse IgG2a, but not Mouse IgG1. Refer to the Protein A and Protein G afnity information and prod-

uct insert sheets to identify the correct afnity resin for a particular type of antibody capture. IMAC afnity resin is used to

capture His-tagged recombinant proteins. The sample pH must be correct – in most cases, a sample of pH 7 is desirable

for capture. For some sample proteins, it is important to increase the tip bed residence time to compensate for low-afnity

interactions. Capture residence time is increased by decreasing capture ow rate and/or increasing the number of capture

cycles.

What are the afnities of Protein A and Protein G for any specic protein?

Protein A afnity resin specically binds to most IgG’s with high afnity. However, in many cases, Protein G afnity resin

may be useful or even necessary. For example, mouse IgG1 is captured more effectively by Protein G resin over Protein

A resin. Refer to the Pro A and Pro G Afnity information sheets for reference tables of antibody afnities or talk to Rainin

customer support for more details.

What is the maximum loading capacity for a PureSpeed Tip? For example, what is the loading of a Protein A

PureSpeed Protein Tip?

When using the largest bed volume 1000 PureSpeed tips, up to 2 mg of human IgG can bind to the Protein A afnity

resin provided the sample contains enough protein and proper loading procedures have been followed. To fully load the

resin bed, a slight amount of excess protein is needed in the sample. High loading is achieved with slower ow rates

or more loading cycles; however, if the sample protein excess is large, ow rate and number of loading cycles are less

important.

What size proteins can I purify with the PureSpeed system? For example, can I purify protein complexes that are

very large?

Most researchers study proteins in the 5 to 200 kDa size range. Interestingly as the protein size increases, the larger,

more bulky proteins may not stick as tightly to the afnity resin and the conditions for purication are usually less strin-

gent to prevent loss of protein. Nevertheless, these proteins are puried quite easily.

Protein complexes however are much larger (e.g. 1 MDa to perhaps the 8 MDa range). Proteins in a complex held in a

strong core of proteins (proteins tightly associated with each other) are usually captured by packed bed columns and will

also be captured by the PureSpeed Protein Tips. Transient (non-core) proteins are often difcult to isolate in a packed bed

system; they are often too large and fragile to survive the process of owing through the packed bed. The gentle action

of the PureSpeed system may enable purication of transient proteins in protein complexes. But each system should be

examined on a case-by-case basis.

What are afnity tags?

Recombinant proteins are generated by introducing an extrachromasomal DNA vector (plasmids containing the gene

of interest) into cells. The plasmid vector then utilizes the cell’s machinery to express the gene to produce the protein of

interest. Afnity tags attached to the N- or C-terminus of the protein are expressed along with the recombinant protein.

These afnity tags make it possible to perform afnity purication with the appropriate afnity resin. After capture and

column washing, the recombinant protein is recovered by eluting the tip with a buffer solution. In some cases, tags can

be enzymatically cleaved of the tip. An example of this is engineering TEV sites into the DNA vectors, in addition to DNA

expressing the tag. The afnity resin will capture the protein with the tag while leaving the TEV site available for cleaving.

Some tags can be attached to proteins directly (without plasmid vectors). An example of this is the chemical attachment

of the biotin tag to lysine residues on the sample protein.

What are some common afnity tags and their counter ligands?

6-Histidine peptide residue (6-His) tag will bind to IMAC afnity resin containing divalent metals including Ni(II) or Co(II).

Glutathione S transferase (GST) tag will bind to glutathione (GST) afnity resin. Biotin tag will bind to Streptavidin afnity

resin. FLAG tag will bind to anti-FLAG antibody afnity resin. Maltose Binding Protein (MBP) tag will bind to amylose af-

nity resin. Myc tag binds to Anti-Myc antibody afnity resin. Calmodulin Binding Protein (CBP) tag binds to Anti-calmod-

ulin binding protein antibody afnity resin. HA peptide tag binds to Anti-HA antibody afnity resin.

What are the major challenges to afnity purication?

Afnity purication methods often struggle to maintain or increase protein concentration while, at the same time, assuring

the protein is pure and active. This is especially true as the amount or volume of protein being puried is decreased. It

can be very difcult to manipulate microliter volumes of sample. Finally, it is difcult to routinely purify large numbers

of samples in parallel in a laboratory environment. PureSpeed technology is designed to easily overcome all these

challenges.

Getting Started: Sample and Tip Prep and Purication Method Design

Why are PureSpeed Protein Tips shipped with some uid in them? What is the uid?

PureSpeed Protein Tips are delivered with glycerol shipping uid in order to keep the resin bed hydrated, which is neces-

sary for optimal performance. The PureSpeed Protein tips can be used straight out of the box without preconditioning.

However, the rst step of a programmed method is usually an equilibration step followed by the capture of the protein of

interest.

Is it necessary to condition the PureSpeed Protein Tips before the capture step?

It is not mandatory but recommended in most cases. The purpose of the equilibration step is to dilute the glycerol that is

used to pack the resin in the tips and to make sure the pH of the resin/solution is prepared and optimal for the subsequent

steps.

How clear or clean should my sample be before I begin to use a PureSpeed Protein Tip?

PureSpeed Protein Tips have been used with a variety of different sample media. In all cases we recommend that the

solution to be puried is clear of any particulate substances. In order to guarantee this, the sample should be centrifuged

then the clear supernatant processed by the tip. Care should be taken not to disturb the plug of spin down residue while

sample is processed. After centrifugation, it is recommended to transfer the sample to a fresh well before processing.

What is the maximum sample volume I can process with my PureSpeed Protein Tip?

For the 1000 tips we recommend a maximum of 1 mL sample and for the 200 tips a maximum of 200 µL sample.

Larger samples can be processed by aliquoting sample into multiple wells of a plate, which are then processed se-

quentially. For example, a researcher purifying 5 mL of sample should perform 5 separate capture steps. First, aliquot

the protein sample into ve separate 1 mL volumes, then program the pipette to perform a capture step on each of the

individual aliquots. In this way, there is a maximal capture of all of the protein in the original 5 mL. The subsequent wash

and elution steps can proceed as normal.

Protein Recovery

If my antibody is very dilute (e.g. 5 µg/ml) and I have 5 mL of solution, which PureSpeed Protein Tips do you recom-

mend and how do I recover as much protein as possible?

Using the 1000 PureSpeed Protein Tips with the 20 µL bed, divide the 5 mL sample into 5 equal volumes of 1 mL.

Perform the Capture step for at least 2 cycles (preferably 4 cycles) sequentially for each 1 mL of sample. After tip

washing, elute the protein captured with 3x the resin bed volume of elution buffer. In an alternate capture procedure, the

sample could be put into a 6+ mL vial and the entire sample processed. In this case, only a portion of the sample can

travel through the tip in any one cycle. Approximately 15 cycles should be used to ensure that the sample protein travels

through the tip at least once and is captured. After sample capture, process the PureSpeed Protein Tip in the normal man-

ner as described above. The rst sequential procedure is preferred.

I followed all of the instructions for isolating my protein, but I see nothing on my SDS-PAGE gel. What do I do now?

Check for the presence of your protein in the capture ow-through solution using SDS-PAGE. If none is present, then there

are only two possibilities: either the sample has not expressed and there is none to be captured, or the sample did ex-

press and was captured but did not elute from the tip. Highly expressed samples can sometimes be detected directly on

the SDS-PAGE. Assuming that sample has expressed, conrm that the sample capture pH is correct (approximately 7.4)

and that the sample is compatible with the tip chemistry being employed. Also, increase the elution strength with parallel

experiments to study the effect on recovery. Many users use a known standard protein with known characteristics. The

sample and standard are processed in parallel whenever a new unknown protein is being expressed and/or puried for

the rst time.

I am using the PureSpeed ProA tips to isolate Mouse IgG, but I don’t see any bands in my gel. How do I know that

my system is working?

Review the expression system and the type of mouse IgG in the sample. Refer to Protein A and Protein G Afnity informa-

tion sheets for reference tables of antibody selectivities for Afnity resins. Check the ow-through from your capture steps

to verify that the protein was captured. Check the wash conditions to make sure the specic protein of interest was not

removed in the wash step. Next, check to see if the protein is still attached to the resin after the nal elution step (pH of

buffer may not have been low enough). Finally, check to see if the eluted protein neutralized effectively and did not dena-

ture. Many users run an antibody standard in parallel with the sample to help monitor loading and recovery performance

of the sample.

What if I put excess protein through the PureSpeed Protein Tip. Does that affect my ability to capture the protein I

want?

No, once all capture sites are full, excess protein does not affect the existing captured proteins. This process will produce

an extremely concentrated puried protein when the sample is nally recovered in the small elution volume.

What is the maximum mass that I can get from a 20 µL ProA, ProG or IMAC tip? What about other bed volume tips?

The PureSpeed process can often capture more protein onto the resin bed than suggested by the original resin manufac-

turer. The repeat cycling of the sample capture process used by the PureSpeed maximizes resin performance. Multiply the

resin capacity per unit volume by the number of µL in the tip bed.

Can I recover more material if I do two elutions rather than one?

Experiments suggest that with a single elution step (and depending upon the afnity of the resin and the elution strength

of the elution buffer) anywhere between 60 and 80% of the puried protein can be removed. This percentage can be

increased with a second or third elution step if the end goal is to recover more protein. But this will be at the expense of

concentration if the elution volumes are combined.

How do I know that all of the protein has been eluted from the resin?

Elution of protein from the tip can produce two different end results. If the requirement is for highest concentration then

the minimum volume of elution is recommended. However, this may result in a lower percentage of puried protein be-

ing removed from the tip. If the goal is maximum mass of puried protein then several wash steps will ensure maximal

removal of protein from the resin. However, this will reduce the concentration of nal protein.

It is unlikely that 100% of protein will be removed from the resin and researchers can expect that, with sufcient elution,

approximately 95% of protein can be removed from the resin.

I heard that you can elute from an IMAC resin using variable pH. Is this true?

Yes, low pH elution buffer can remove a His-tagged protein from an IMAC resin. In addition, adding a chelator such as

disodium EDTA to the elution will not only remove the protein from an IMAC tip, but the nickel metal as well.

Protein Purity

My IgG protein doesn’t seem to be pure. I expected to see only one band but I get two on the gel. What’s wrong?

One would see two bands on SDS-PAGE: a light chain and heavy chain. A single band is expected if the sample has not

been treated to break the sample into the 2 chains.

I am running an IMAC resin and my nal product is not as pure as I had hoped. How can I improve that?

Wash buffers for IMAC resin will often contain low levels of imidazole that helps to wash away some of the non specic

2, 3, 4 or 5 His-tagged variants that may have been expressed along with the specied 6-His product. Imidazole in the

wash helps remove any histidine rich protein in your sample that competes for Ni(II) in the resin. Sometimes the con-

centration of the imidazole is not high enough to remove these contaminants (the wash is not stringent enough) so the

nal product will not appear as pure as it could be. But care must be taken if the wash buffer strength or stringency is

increased because the sample protein of interest might also be removed in the wash. If the wash stringency is too high,

the protein might be very pure, but the yield might also be very low.

Why do you recommend two different types of wash buffers while using ProA or ProG resin? What is the second

buffer and what does it do?

Two wash buffers enhance the elution step by reducing the pH shock when moving from a pH 7 wash buffer to a pH 2

elution buffer. Low pH elution buffer has higher strength to break the binding between resin and protein and can improve

protein recovery. If there is no intermediate saline wash, the residual pH 7 wash buffer that resides in the resin will de-

crease the pH strength of the elution buffer, which then results in a lower yield of eluted protein. Adding an extra step to the

process removes the buffering effect of the rst wash step and enables the low pH of the nal elution step to be effective

and produce the highest recovery of puried protein. The second wash can be very important to achieving reproducible

elution of the protein and is always recommended.

What is the minimum recommended wash buffer volume that still achieves the highest purity of sample?

This will depend upon the volume of the resin in the tip, the type of protein being puried and the types of contaminants

that are mixed in with the protein of interest. For example, a 200 µL wash of a 5 µL resin bed will probably produce the

optimal purity results. It is possible that the same level of purity could be achieved using a 100 or even a 50 µL wash

volume. However, given the cost of the wash buffer and the difference in time between a 50 and a 200 µL wash, the

recommendation is to go for the highest volume rather than try to compromise.

Is there imidazole in the equilibration buffer and sample buffer? If yes, why?

We recommend using the following equilibration buffer while using IMAC resin: 50 mM sodium phosphate, 1.5 M

sodium chloride, 25 mM imidazole, pH 7.4 Note: This is a 5X buffer and needs to be diluted to 1X with water before use.

Imidazole is present to clean the tip of any material that might bind to Ni on the tip. Imidazole in the sample buffer can

help keep the protein pure by limiting the capture of impurities along with the recombinant sample protein.

What is the difference between high and low stringency buffers?

Low stringency washes remove weakly bound non-specic impurities. If the sample protein is large and bulky, then the

tip should be washed with low stringency buffers to avoid losing sample protein. Higher stringency washes can be used

for tightly held sample proteins. For IMAC resins, the difference between the low and high stringency buffers is the con-

centrations of imidazole used. For IMAC, a low stringency buffer is 5 mM imidazole and a high stringency was is 20 mM

imidazole. For ProA/ProG resins, the stringency of the wash can be controlled by the volume of the wash. Smaller wash

volumes provide a low stringency wash. Large wash volumes provide a higher stringency wash. Stringency for these

resins can also be controlled by the wash pH (lower pH is a higher stringency wash), but this should done carefully with

testing to avoid sample protein loss.

Protein Activity

Why do I need the neutralization buffer?

If low pH elution is used, the protein may denature if left at low pH for an extended period of time. Although there is no set

time period, the protein buffer pH should be raised to neutral pH in an expedient manner. The neutralization buffer is con-

centrated and can be added directly to the eluted protein to raise the pH to neutrality. When necessary, conrm that the

pH has been raised to 7 after an aliquot of the concentrated neutralization buffer has been added to the eluted sample.

Will my puried protein still be active or will it denature?

PureSpeed Tips have an ultra low non-polar surface area to prevent denaturation of proteins. Many different protein types

have been puried and enriched using PureSpeed Protein Tips, including membrane proteins. PureSpeed puried proteins

likely maintain their activity during and after the purication process.

What is the optimal elution pH for a Protein A or Protein G tip? How quickly should I neutralize the eluted protein?

How should the eluted protein be neutralized?

The optimal pH will vary depending upon the protein that’s captured and the afnity it has for the resin. Smaller proteins

bind tighter to the resin. Obviously, the most gentle conditions are desired for elution. Most proteins will elute from the

resin at pH 2.5 – 3.5, but it may be necessary to elute as low as pH 2.0. Contact Rainin for the types of low pH buffers

used for elution. After elution, neutralization with a buffer is recommended to bring the pH back up to neutral as soon as

possible – no longer than a few minutes. The neutralization buffer may be a pH 8 or 9 depending upon the strength of the

buffer used to elute the protein. See the protein purication manual and buffer recipe documents for details.

Optimization and Enhancement of the Purication Process

1. What basic parameters can be optimized and enhanced in the PureSpeed tip process?

In general, excellent results are obtained with the PureSpeed tip using the default methods contained within the E4 XLS

pipette software. However, higher performance results can be obtained depending on the goals of the researcher. Param-

eters that can be optimized or enhanced include concentration, mass, purity or activity of the recovered protein, the speed

of the purication process and the ability to accommodate various starting sample volumes.

2. Why and how should the capture step be enhanced?

In order to obtain optimum recovery, whether it is concentration or mass amount, it is rst necessary to make certain

the capture is optimized and as much protein is captured onto the resin bed as possible. Of course, this starts with the

expression of the protein – it can’t be captured if it isn’t there in the rst place. The best way to measure expression of a

protein is to run a control protein in parallel with the sample under exactly the same conditions.

Parameters that increase capture include increasing the number of capture cycles and slowing down the capture ow

rate. In many cases, adding salt to the sample solution will increase uptake. Salt reduces the solubility of the protein

and can increase the selectivity of the resin for the protein. These salts enhancing protein selectivity for the resin follow

the Hofmeister series: increased selectivity effect of anion to decreased selectivity: PO

> SO

> COO

> Cl

; increased

selectivity effect of cation to decreased selectivity: NH

> K

> Na

. Also, it is important to check the sample pH – uptake

of protein is optimized at neutral pH for most afnity resins.

3. Why and how should the elution step be enhanced?

Removal (elution) of the protein from the resin in the PureSpeed tip can be surprisingly difcult for some sample proteins.

Running a control protein in parallel with the sample under exactly the same elution conditions is an excellent measure of

elution efciency, provided the control protein is similar in structure to the sample protein. Large bulky proteins tend to be

easy to elute from the tip whereas smaller proteins are more difcult.

Standard elution volumes are 3x of the resin bed volume. A higher concentration of protein can be achieved by reducing

the elution volume to 2x of the resin bed volume. Many researchers will perform a second elution and combine the frac-

tions. This will increase mass recovery but reduce the overall concentration of the recovered protein.

4. How can the highest concentration of recovered protein be achieved with all protein in the sample recovered?

Use a tip with sufcient capacity to capture the entire amount of protein. Passing the sample over the resin bed several

times allows as much capture of the protein from the sample as possible. Using a slower capture ow rate also increases

the residence time of the sample in the resin bed which increases the capture of the sample. In cases where the sample

volume is signicantly larger than the total volume of the PureSpeed tip, use more capture cycles rather than a slower

ow rate so that eventually all of the sample protein passes though the resin bed and is captured. Better still, the sample

volume can be split into several volumes and each sample volume captured in succession.

5. How can the highest concentration of recovered protein be achieved?

In some cases the type of resin can be important. For example, Pro A resin has a higher capacity for many types of

antibodies than Pro G resin. Also, use sufcient sample target protein so that all of the resin’s functional groups have

captured protein attached. If necessary, follow all of the optimization procedures for the number of capture cycles and

ow rate. Finally, using a smaller elution volume increases the concentration of the eluted protein.

6. How can high normalized concentration of recovered proteins be achieved?

Recovering a normalized concentration of protein (the concentration of protein recovered from the tip is uniform regard-

less of the starting sample concentration) can be useful in many cases. Many assays, especially cell based assays,

require a minimum and uniform amount of puried protein. This can be difcult to achieve if the expression efciency or

effectiveness of the protein is either unknown or known to vary for a series of proteins.

PureSpeed tips are unique in their ability to normalize concentration of puried protein to a predictable level. Starting

sample protein concentrations that vary widely can still be brought to a uniform concentration in the puried form. The

resin bed is overloaded even with the lowest expressed protein by using sufcient sample concentration and volume.

The rst step is to use the smallest possible bed volume that is appropriate for the assay. In most cases, this will be the

5 µL bed volume. Overload the resin bed by a factor of two or more. If the concentration of a sample or series of samples

is unknown, it is best to process enough sample volume so that the lowest expected expressed sample has sufcient

volume to overload the tip, then treat all samples in the same way. It may be necessary to process several aliquots of the

sample in series to overload the tip.

7. How can the highest concentration of puried protein suitable for structural analysis be achieved?

Several parameters are important. High concentration is important of course, but the recovered protein must also be in a

suitable buffer environment for forming crystals and have the correct folding and protein activity. Therefore it is necessary

to follow the guidance above, but also perform the operations in parallel to screen different buffers.

A two-step process for achieving ultra-high protein concentration is possible with the PureSpeed tip but is beyond the

discussion within this Guide. Contact Rainin for more details.

8. How can the highest mass amount of recovered protein be achieved?

Use the largest resin bed volume so that the resin bed is not overloaded (there is no protein in the capture ow through).

The capacity of the resin bed is proportional to the bed volume. If the sample volume is large, be sure to process (with

sufcient cycling) an aliquot of sample completely with each capture cycle, then repeat the cycling process with another

aliquot until the entire sample is processed. Another strategy is to process only part of the total volume with each capture

cycle, but use several capture cycles. Eventually the sample protein will travel through the bed and be captured. Typically

10 or more capture cycles is used in this strategy. If the resin bed capacity is severely overloaded two PureSpeed tips can

be used. Generally two elution aliquots should be used to recover the entire amount of protein from the resin bed.

9. How can the highest purity of recovered protein be achieved?

Use progressively higher concentrations of wash solvent to remove non-specic bound protein and check the purity of

recovered protein using gel electrophoresis. The wash can remove the captured protein as well as non-specic bound

proteins and other materials. This is especially important in PureSpeed tip extractions because the ratio of wash solvent

volume to bed volume is extremely large. So this process should be done carefully using only lowest concentration of

solvent necessary for cleaning. At least two aliquots of wash solvent are necessary; more aliquots may be used but usu-

ally are not necessary.

10. How can the highest activity of recovery protein be achieved?

It is important that activity of a protein be retained and not denatured. In that respect, it is important to use buffer condi-

tions that are not denaturing. Generally this means that the buffer pH is kept neutral and at high ionic strength. Many

researchers purify in parallel and screen expression conditions and purication conditions. Activity can be tested with

enzymatic assays, ELISA, SPR or a functional test.

11. How can the purication process be sped up?

One answer is to process as many samples in parallel as possible. Of course reducing the number of cycles will reduce

time. If possible, reduce the wash and elution/enrich cycles before the number of capture cycles is reduced. Often, for

washing, usually only 1 cycle of each solvent is adequate. Use fewer capture cycles or use a fast ow rate, but only if

necessary. Reducing the number of capture cycles to 2 is acceptable in almost all cases, but could reduce the amount of

protein captured and increase the deviation of the recovery from sample to sample.

12. How can the smallest sample volumes be processed?

The ability to process small sample volumes enables the possibility of the expression of small volume of proteins, reduc-

ing the amount of material required to produce the desired protein. Normal sample volumes range from 0.2 mL to 1 mL,

but samples can be as large as several mL or as small as a few µL. Because of the low dead volume and low resin bed

volume of the PureSpeed tips, very small elution volumes can be used to elute protein from the resin bed. Very high pro-

tein concentrations are achieved with these extremely small elution volumes. The concentration of the protein increases by

using decreasing elution volumes (although less mass amount of the protein may be recovered). Use the smallest resin

bed volume possible and use the smallest elution volumes (the concentration increases with decreasing elution volume).

13. How should large sample volumes be processed?

Two different methods are normally employed. Sample volumes larger than the tip volume can be processed simply by

inserting the tip in the sample and processing with sufcient number of cycles so that any sample protein in the sample

has a chance to contact the resin bed. A general rule is to double the number of capture cycles for every tip volume of

sample. Thus, if a 1 mL sample in the 1000 tip (with a 1 mL tip volume) uses 4 capture cycles, then a 2 mL sample

using the same tip would require 8 capture cycles, a 3 mL sample,16 capture cycles, and so on. A better method is to

split the sample into as many aliquots as necessary and process each aliquot in succession until the entire sample is

captured. Then, proceed as normal with the wash/purication step and the elution/enrichment step.

Rainin Instrument, LLC

7500 Edgewater Drive, Box 2160, Oakland, CA 94621-0060

+1 510 564 1600

www.mt.com/rainin

a METTLER TOLEDO Company

9920-411G Guide Rev A

Mettler Toledo PureSpeed Operating instructions

Mettler Toledo PureSpeed Operating instructions

Mettler Toledo PureSpeed Operating instructions

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