PageBlue™ Protein Staining Solution

- Store at room temperature

Catalog#	Size, concentration	Certificate of Analysis	MSDS

R0571	1 liter (for up to 150 mini gels)	R0571

Product information

PageBlue™ Protein Staining Solution

Features

Ready-to-use.
Easy-to-use – simply soak the gel in the solution and observe stained bands. No destaining required.
High sensitivity – 5 ng of protein can be detected. More sensitive than Coomassie Brilliant Blue R-250.
No destaining required.
Safe – does not contain methanol or acetic acid.
Broad linear dynamic range.
Fast staining – 25-40 minute protocol.
Cost efficient – can be reused up to 3 times.

Applications

Visualization and quantification of proteins separated in 1D, 2D, IEF polyacrylamide gels.
Protein staining after transfer onto PVDF membranes.
In situ zymography.
Compatible with mass spectrometry, silver staining.

Description

PageBlue™ Protein Staining Solution is a ready-to-use solution for fast and sensitive staining of proteins separated in polyacrylamide gels or PVDF membranes. PageBlue™ is based on the Coomassie Brilliant Blue G-250 dye.

PageBlue™ dye forms colloidal particles allowing proteins to be preferentially stained eliminating the need for laborious, expensive and hazardous gel de-staining procedures. Proteins are stained to an endpoint, therefore over-staining does not occur even after overnight staining.

The linear dynamic range of PageBlue™ extends over two orders of magnitude (5-500 ng, see picture below) and is about 10 times more sensitive than traditional Coomassie Brilliant Blue R250. PageBlue™ staining solution does not contain methanol or acetic acid and can be reused up to three times without any loss in sensitivity.

Quality Control

Tested in staining of the Protein Molecular Weight Marker on SDS polyacrylamide gel.

Storage

Stable at 4 to 26°C.

Figure 1. Sensitivity and linear dynamic range of PageBlue™ Protein Staining Solution.
A – beta-galactosidase
B – bovine serum albumin
Electrophoresis conditions: 12% Tris-glycine SDS-PAGE, 0.75 mm gel, well width 3 mm.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Gel Staining Procedure with PageBlue™ Protein Staining Solution

With microwaving (fast protocol)	Without microwaving (conventional protocol)
Total time
25 min for native gels 40 min for SDS-containing gels	65 min for native gels 95 min for SDS-containing gels
*1. Washing in water. Repeat 3 times**
Add 100 ml water Microwave for 1 min Wash with gentle agitation for 5 min Discard the water	Add 100 ml water and rinse Discard the water
2. Staining
Add 20 ml PageBlue™ Protein Stainin Solution Microwave for 30 s Stain with gentle agitation for 20 min Discard the solution	Add 20 ml PageBlue™ Protein Staining Solution Stain with gentle agitation for 60 min (or overnight) Discard the solution
3. Washing in water
Add 100 ml water and rinse for 5 min	Add 100 ml water and rinse for 5 min

* only SDS-containing gels

Note

PageBlue™ Protein Staining Solution can be reused up to 3 times without a decrease in sensitivity.
All reagent volumes are for 8x10 or 10x10 cm minigels of 0.75-1 mm thickness. Gels should be completely immersed in solution.
When several gels are being stained, increase the amount of staining solution accordingly.
The first wash step is crucial to remove SDS from the gel as SDS interferes with the staining reaction.
For staining native gels without SDS, the washing step is not required.
Staining sensitivity can be increased if the proteins are fixed for 15 min either with 12% trichloracetic acid or with 25% isopropanol supplemented with 10% acetic acid. Fixation prevents protein diffusion from the gel and accelerates SDS removal. After fixation, gels can be stained immediately without additional washing.
Using either the fast or conventional protocol, staining sensitivity is 5 ng of protein per band. To increase sensitivity to 0.05 ng per band the gel can be stained using the PageSilver™ Silver Staining Kit.
For staining peptides or small proteins (more than 10 kDa) fixation of the proteins for 15 min either with 12% trichloracetic acid or with 25% isopropanol supplemented with 10% acetic acid is recommended. Fixation prevents protein diffusion from the gel and accelerates SDS removal. After fixation, gels can be stained immediately without additional washing. Overnight staining time is required for peptide detection.

Staining PVDF Membrane with PageBlue™ Protein Staining Solution

Note

PVDF membrane must be air-dried before staining.

Add PageBlue™ Protein Staining Solution to cover the PVDF membrane. Agitate gently for 2 min.
Wash with 30% ethanol with gentle agitation for 5 min.

To completely remove the stain, wash the membrane with the mixture of 30% acetonitrile and 20% ethanol for 5 min.

Staining of Small Proteins

Protein fixation with glutaraldehyde is required before staining the gel with Coomassie Brilliant Blue dye (PageBlue™ Protein Staining Solution, #R0571) or silver (PageSilver™ Silver Staining Kit, #K0681).

Note

Other compounds commonly used for protein fixation (e.g., acetic acid, isopropanol, ethanol, TCA) are not suitable; proteins will wash away during staining procedure.

Procedure

Add 100 ml of deionized or distilled water to the gel and wash for 1 min with gentle agitation. Discard the wash.
Add 50 ml of freshly prepared 5% glutaraldehyde solution (gel should be covered completely). Fix with gentle agitation for 30 min. Discard the solution.
Add 100 ml of deionized or distilled water to the gel and wash for 5 minutes with gentle agitation. Discard the wash. Repeat this step twice. The gel is now ready for staining.

For detailed staining protocols using Fermentas PageBlue™ Protein Staining Solution or PageSilver™ Silver Staining Kit, refer to the product manuals.

Note

Proceed directly to the staining step in PageBlue™ Protein Staining Solution protocol and to sensitizing step in PageSilver ™ Silver Staining Kit protocol.

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