In Western blots, proteins are transferred from polyacrylamide gels to membranes. Total protein stains can be useful to assess the efficiency of this process. Staining the membrane confirms protein transfer, while staining the gel reveals any proteins that have not transferred.
What information can I gain from staining?
Staining can reveal valuable information:
- Irregular staining patterns across the gel and membrane suggest transfer has been uneven.
- Little or no staining on the membrane indicates that transfer has not occurred.
- Variation in band intensity between lanes suggests unequal loading of sample.
- Possibility of quantitative comparison between protein bands of interest.
In addition, standards with known molecular weights can indicate if large and small proteins have transferred with the same efficiency
Prestained, multicolored molecular weight markers (Table 1) can be used to track progress in electrophoresis and transfer. In addition, colored markers containing tagged proteins are available for accurate protein size determination and for monitoring transfer efficiency in enhanced chemiluminescent (ECL) and fluorescent detection systems.
Table 1. Prestained and fluorescent protein markers for tracking progress of electrophoresis and transfer of proteins to Western blots
|Product||Quantity||Molecular weight range (Mr)||Description|
|Amersham ECL Rainbow Marker – Full Range||250 µL||12 000 to 225 000||Ten separate proteins with six different visible colors. Monitor progress of protein electrophoresis and assess transfer efficiency and molecular weight of blotted proteins without post-staining.|
|Amersham ECL Rainbow Marker – High Range||250 µL||12 000 to 225 000||Eight separate proteins with six different visible colors. Monitor progress of protein electrophoresis and assess transfer efficiency and molecular weight of blotted proteins without post-staining.|
|Amersham ECL Rainbow Marker – Low Range||250 µL||3500 to 38 000||Seven separate proteins with five different visible colors. Monitor progress of protein electrophoresis and assess transfer efficiency and molecular weight of blotted proteins without post-staining.|
|Amersham ECL DualVue Marker||25 loadings||15 000 to 150 000||Detect proteins on gels, membranes, and film. Three bands visible on gels and blotting membranes and seven bands visible on chemiluminescence images.|
|Amersham ECL Plex Rainbow Marker||120 to 500 µL||12 000 to 225 000||Protein molecular weight marker bands visible on gels, membranes, and on fluorescence images using Cy3 and Cy5 channels.|
|Amersham QuickStain||500 µL||12 000 to 225 000||Cy5 fluorophore and labeling buffer for fast detection of proteins in PAGE gels or directly after transfer to Western blots. Eliminates the need for post-staining with Coomassie or silver stains.|
Selecting a total protein stain
As discussed, efficient protein transfer enables accurate quantitation and analysis in Western blotting. Total protein staining can identify problems in the transfer process, allowing the user to modify the protocol and rectify the situation.
Key considerations when choosing a stain are sensitivity and the potential for interference with antibody binding. Other considerations include compatibility with gels and membranes, staining times, cost, and safety issues.
Table 2 summarizes the advantages and disadvantages of common total protein stains.
Table 2. Commonly used total protein stains
|Total protein stain||Advantages||Disadvantages|
|Colloidal gold||Compatible with nitrocellulose (NC) and PVDF membranes||Can take longer to develop than other methods|
|Highly sensitive (1 ng 1 /band)|
|Signal can be boosted with enhancement reagents|
|Coomassie™ Brilliant Blue||Inexpensive||Proteins might release the dye during background staining|
|Reusable||Proteins destain at different rates|
|Compatible with gels and membranes||Time consuming|
|Sensitive (50 to 100 ng1)||Relatively narrow dynamic range|
|Ponceau S||Compatible with NC and PVDF membranes||Poor sensitivity (> 250 ng)|
|Rapid, easy to use||Incompatible with SDS|
|Stable in storage||PVDF membranes require prewashing in methanol because of incompatibility with sodium dodecyl sulfate (SDS) in SDS-polyacrylamide gel electrophoresis (PAGE)|
|India Ink||Rapid||Poor contrast in photographs|
|Inexpensive||Pretreatment with blockers is necessary to reduce nonspecific staining|
|In solution stable for 1 mo (month)||Irreversible|
|Amido Black||Rapid||Membrane distortion risk|
|Stable||Not compatible with immunodetection|
|Sensitivity of 50 to 100 ng1|
1 Protein quantities expressed as ng/band in gel
To find out more about protein staining in Western blotting, see the principles and methods handbook.