Thermo Fisher Scientific Pierce Phosphoprotein Staining Kit User guide

Brand: Thermo Fisher Scientific

Model: Pierce Phosphoprotein Staining Kit

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

24550 Pierce Phosphoprotein Staining Kit, contains sufficient materials to stain 10 (8 × 8cm) gels − kit

includes the Pierce Phosphoprotein Stain Reagent Set (Product No. 24551) and Phosphoprotein

Control Set (Product No. 24552)

24551 Pierce Phosphoprotein Stain Reagent Set

Reagent 1: Sulfosalicylic Acid Solution, 1000mL

Reagent 2: Sulfosalicylic Acid + CaCl2 Solution, 250mL

Reagent 3: 0.5N NaOH, 250mL

Reagent 4: Ammonium Molybdate Solution, 500mL

Reagent 5: Ammonium Molybdate + HNO3 Solution, 250mL

Reagent 6: Methyl Green Solution, 250mL

Reagent 7: 7% Acetic Acid, 500mL

24552 Pierce Phosphoprotein Control Set

Positive Control (Phosvitin), 1mg (~40kDa in gel)

Negative Control (Soybean Trypsin Inhibitor), 1mg (~20kDa in gel)

Storage: Upon receipt store the control set at 4°C. Store all other reagents at room temperature.

Product is shipped at ambient temperature.

Introduction

Protein phosphorylation and dephosphorylation play significant regulatory roles in a variety of cellular processes such as

normal and abnormal cell growth, cell death and secretion. The Thermo Scientific Pierce Phosphoprotein Staining Kit

specifically stains phosphorylated proteins directly on gels. Staining is achieved by first hydrolyzing the phosphoprotein

phosphoester linkage using 0.5N NaOH in the presence of calcium ions. The gel containing the newly formed insoluble

calcium phosphate is then treated with ammonium molybdate in dilute nitric acid. The resultant insoluble nitrophospho-

molybdate complex is finally stained with the basic dye, Methyl Green Solution. The reagents in this kit hydrolyze the

phosphoester linkage of phosphoserine and phosphothreonine. Phosphotyrosine is not hydrolyzed and cannot be detected

with this kit.

The limit of detection of this stain depends on several factors, including the molar amount of phosphate loaded onto the gel

and the accessibility of these groups within the electrophoresed sample to hydrolysis during the staining procedure. For

several proteins, the calculated lower molar limit of phosphate detection varied more than five-fold (Table 1) and, therefore,

lower limits of detection must be determined empirically for each phosphoprotein.

Preparation of Controls

1. Dissolve contents of each phosphoprotein control vial in 1mL Tris-buffered saline (e.g., 25mM Tris, 180mM NaCl;

pH 7.2; Product No. 28376) to yield 1mg/mL (1µg/µL) stock solutions.

2. Prepare a 10-fold dilution to yield a 0.1µg/µL working solution. To ensure appropriate sensitivity of kit and detection

range for samples, load several concentrations (e.g., 10, 1 and 0.1µg) of the positive control

3. Mix and heat controls with the appropriate volume of gel sample loading buffer and load 2-10µL onto the gel lanes.

0912.2

24550 24551 24552

Pierce™ Phosphoprotein Staining Kit

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Procedure

Note: To avoid background, do not touch the gel throughout the procedure. Perform the entire procedure in one staining tray

by decanting one reagent (without touching the gel) and then adding the next. Use sufficient reagent volumes to ensure that

the gel is submerged during each incubation.

1. Pre-heat an incubation oven to 65°C in preparation for Step 6. Do not use a water bath for this step because water baths

produce poor results.

2. After electrophoresis, place the gel into a tray, add 50mL of ultrapure water and wash the gel with mild agitation for

10 minutes.

3. Discard the water, add 25mL of Reagent 1 and gently agitate the gel for 15 minutes.

4. Discard Reagent 1, add 25mL of Reagent 2 and gently agitate the gel for 30 minutes.

5. Discard Reagent 2 and quickly rinse the gel with ultrapure water to remove any surface CaCl2.

6. Discard the rinse water; add 25mL of Reagent 3, cover the tray and place it in a 65°C oven for 20 minutes. There is no

need to agitate the gel during this step.

Note: The gel will swell to approximately 110% of its original size in this step. Use a tray large enough to accommodate

this increase. The tray containing the gel must be covered to prevent evaporation and uneven wetting to the gel, which

can result in high background. The formation of a white precipitate is normal during this step.

7. Discard Reagent 3, add 25mL of Reagent 4 and gently agitate the gel for 10 minutes. Repeat this step once.

8. Discard Reagent 4, add 25mL of Reagent 5 and gently agitate the gel for 20 minutes.

9. Discard Reagent 5, add 25mL of Reagent 6 and gently agitate the gel for 20 minutes.

10. Discard Reagent 6, destain the gel by adding 25mL of Reagent 1 and gently agitating the gel for 15 minutes. Repeat this

step once. At this stage, a green band of phosphoprotein can be visualized against a faint green background.

11. Completely destain the gel in 25mL of Reagent 7 and incubate overnight with gentle agitation. Change the solution once

after it becomes green in color.

Note: The gel can be dried after first incubating it in drying solution (5% glycerol and 10% ethanol in water) for at least

5 hours. The gel also may be stained for total proteins using Thermo Scientific GelCode Blue Stain Reagent (Product

No. 24590) after washing it three times with 100mL of ultrapure water for 10 minutes each.

Table 1. Lower limits of detection for several phosphoproteins using the Thermo Scientific

GelCode Phosphoprotein Stain) after washing it three times for 10 minutes in 100mL of

ultrapure water.

Protein

Phosvitin

β-casein

Ovalbumin

Histone Type III-S

Lower limit of detection

(total phosphoprotein) 0.080µg 0.160µg 10.0µg 10.0µg

% Phosphate

(by weight) 10% 1% 0.12% 0.02%

Molar ratio

(phosphate:protein) 100:1 4.39:1 1.73:1 0.26:1

Lower limit of detection

(weight of phosphate) 8ng 1.6ng 12ng 2ng

Lower limit of detection

(moles of phosphate)

101pmol 20pmol 151pmol 25pmol

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

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