Thermo Fisher Scientific Pierce Biotinylated Recombinant Protein G User guide

Brand: Thermo Fisher Scientific

Model: Pierce Biotinylated Recombinant Protein G

Type: User guide

INSTRUCTIONS

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Number Description

29988 Pierce Biotinylated Recombinant Protein G, 0.5mg, contains 1mM sodium citrate buffer at pH 6.8

when reconstituted with 0.5mL of ultrapure water; any suitable buffer also may be used for

reconstitution

Storage: Upon receipt store product at -20°C. Product is shipped at ambient temperature.

Introduction

Protein G is a bacterial cell wall protein isolated from group G Streptococci. Protein G binds to many mammalian

immunoglobulins mostly through their Fc regions. Native protein G has two immunoglobulin binding sites, as well as sites

for albumin and cell surface binding; however, in the Thermo Scientific Pierce Biotinylated Recombinant Protein G, the

albumin and cell surface binding sites have been eliminated from recombinant protein G (rProtein G, 22.6kDa), which

minimizes nonspecific binding. Typically, binding to Protein G is achieved using 0.1M sodium acetate buffer at pH 5.4;

however, the optimal pH for binding is species dependent.

General Procedure for Western Blotting

A. Materials Required

• Tris-buffered saline (TBS): 25mM Tris•HCl, 150mM NaCl; pH 7.2 (Product No. 28379)

Note: Many IgGs bind to Protein G at physiological pH; however, others, such as rat IgG2a, bind poorly at pH 7 and

significantly better at pH 5.0. Therefore, when necessary, use 0.1M sodium acetate, pH 5.0 instead of TBS.

• Blocking Buffer: TBS containing 1-3% bovine serum albumin (Thermo Scientific Blocker BSA in TBS, Product No.

37520)

Note: A final concentration of 0.05% Tween®-20 Detergent in the blocking buffer often improves blocking; however, it

is not required nor recommended for all systems. Use high-quality products such as Thermo Scientific Surfact-Amps 20

(Product No. 28320), which is a specially purified Tween-20 Detergent free of peroxides and carbonyls that may

interfere in some systems.

• Primary Antibody diluted to the appropriate concentration

• Enzyme-labeled Avidin or Streptavidin diluted to the appropriate concentration with TBS

• Enzyme Substrate: for example, use Thermo Scientific 1-Step TMB-Blotting (Product No. 34018) for HRP

B. Method

Note: Perform incubation steps at room temperature. During each step, use sufficient reagent to cover the entire

membrane. Agitate the membrane when performing the procedure.

1. Incubate membrane, containing the transferred proteins, for one hour in blocking buffer.

2. Rinse membrane three times for 10 minutes each with TBS. Incubate membrane with the primary antibody.

3. Rinse membrane three times for 10 minutes each with TBS. Incubate membrane for one hour with the Pierce

Biotinylated Recombinant Protein G.

4. Rinse membrane three times for 10 minutes each with TBS. Incubate membrane for one hour with the enzyme-

conjugated avidin or streptavidin.

5. Rinse membrane three times for 10 minutes each with TBS. Add the substrate and develop according to the

manufacturer’s instructions.

0094.3

29988

Pierce® Biotinylated

Recombinant Protein G

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

General Procedure for ELISA

A. Materials

• Coating buffer: 0.2M sodium bicarbonate, pH 9.4 (Product No. 28382)

• Phosphate-buffered saline (PBS): 100mM sodium phosphate, 150mM sodium chloride; pH 7.0 (Product No. 28372)

• Blocking buffer: PBS containing 1-3% bovine serum albumin (Blocker™ BSA in PBS, Product No. 37525)

Note: A final concentration of 0.05% Tween-20 Detergent in the blocking buffer often improves blocking; however, it is

not required nor recommended for all systems. Use high-quality products such as Surfact-Amps® 20 (Product No.

28320), which is a specially purified Tween-20 Detergent free of peroxides and carbonyls that may interfere in some

systems.

• Wash buffer: PBS containing 0.05% Tween-20 Detergent

• Primary antibody diluted to the appropriate concentration with PBS

• Enzyme-labeled avidin or streptavidin diluted to the appropriate concentration with PBS

• Enzyme Substrate: for example, use the TMB Substrate Kit (Product No. 34021) for HRP

B. Method

1. Apply antigen solution (150µL of 1-10µg/mL antigen in coating buffer) to each microplate well and incubate for 1 hour

at 37°C or overnight at 4°C.

2. Rinse each well three times with 150µL of wash buffer. Add 150µL blocking buffer to each well, incubate for 30

minutes at 37°C and aspirate.

3. Rinse each well three times with 150µL of wash buffer. Add 150µL antibody solution and incubate for 1 hour at 37°C.

4. Rinse each well three times with 150µL of wash buffer. Add 150µL of Pierce Biotinylated Recombinant Protein G

(1µg/mL) to each well and incubate for 1 hour at 37°C.

5. Rinse each well three times with 150µL of wash buffer. Add 150µL of 0.1µg/mL of the labeled avidin or streptavidin to

each well and incubate for 1-2 hours at 37°C.

6. Rinse each well three times with 150µL of wash buffer. Add the appropriate substrate for detection and develop

according to the manufacturer’s instructions.

Related Thermo Scientific Products

20347 Streptavidin Agarose Resin, 2mL

20357 High Capacity Streptavidin Agarose Resin, 2mL

88816 Pierce Streptavidin Magnetic Beads, 1mL

20227 Pierce Monomeric Avidin Kit

General References

Akerstrom, B. and Bjorck, L. (1986). A physicochemical study of Protein G, a molecule with unique immunoglobulin G-binding properties. J Biol Chem

261:10240-7.

Akerstrom, B., et al. (1985). Protein G: A powerful tool for binding and detection of monoclonal and polyclonal antibodies. J Immunol 135:2589-92.

Akerstrom, B., et al. (1987). Definition of IgG-and albumin binding regions of streptococcal Protein G. J Biol Chem 262:13388-91.

Bjorck, L. and Kronvall, G. (1984). Purification and some properties of streptococcal Protein G, a novel IgG-binding reagent. J Immunol 133:969-74.

Eliasson, M., et al. (1988). Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal Protein G. J Biol Chem 263:4323-7.

Fahnestock, S. (1987). Cloned streptococcal protein G genes. Tibtech 5:79-83.

Sjobring, U., et al. (1988). Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal Protein G. J Immunol 140:1595-9.

Tween is a trademark of Croda International Plc.

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale,

as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and

workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications

regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the

Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This

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