Technique / Proteomics and protein biochemistry / Western blot assay
Western Blot troubleshooting
Western Blot troubleshooting
Western blot troubleshooting tips
- protein transfer problem: poor transfer efficiency, air bubbles trapped when assembling the transfer sandwitch (use a tube or roller to get ride of any trapped air bubbles), voltage and current is not optimized for the size of protein of your interests.
- film development problem: try service and clean the film developer. try film that is optimzed to the signal strength produced by the band of your membrane.
- non-specific bands on western blot: try to use a more specific primary antibody and use optimzed concentration of antibody (in some cases, an used antibody saved from previous run works fine). Blocking is not sufficient and should try overnight blocking at 4c in 5% dry milk (non fat) or BSA solution. Make sure protein lysate is prepared properly without proteolytic degradation (keep lysate on ice or storage in -80c, always use protease inhibitors cocktail).
- High background problem: film exposed too long, too high concentration of secondary antibody, insufficient wash of membrane or strength of detergent in washing buffer.
- Low signal problem: low abundant protein of your interests, primary antibody is not working, secondary antibody too low, washing too stringent. In some cases, a gel documentation system is helpful to capture weak bands. You can also try more sensitive chemiluminescent system which are commercially available from several good vendors.
This troubleshooting guide discuss following problems: (Agrisera)
High background signal
Strange/Non-specific bands on the blot
Low sensitivity
Is a right band detected?
Uneven results with lots of spots
Winning Westerns: 12 Proven Strategies to Optimize Your Western Blots
Last update 26-Jun-2005, Rating of 20 votes.
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By bob
on 21-Mar-2009
I have a prob with transfer, i kept transfer for overnight at 4 degree, still the transfer was not happened, i used 25v 200mA current while transfering, can any one suggest me what exactly time and current will help in transfering, |
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By Rain
on 11-Mar-2009
Dear Friends: |
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By Aye
on 04-Feb-2009
As for the repeat on the western blotting of parafibromin on a DCIS case (+3 intensity), I have sucessfully extracted proteins from the paraffin sections after modifying the extraction process i.e. heat antigen retrieval with 2% SDS for 2 plus hours. According to the datasheet of parafibromin Ab, the expected band size for parafibromin is ~64kDa. However, my Western blot results reflected three bands, two sizes of ~25kDa and one of ~15kDa. Could there be a possible explanation for this? I was suspecting it maybe due to the nature of the protein (e.g. presence of disulphide bonds) or it could be due to the paraffin embedding process and the antigen retrieval using heat. Maybe you can advise me on this. |
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By wilson
on 04-Jan-2009
I have some problem with western currently, my membrane was stripped & incubate with different antibody, the problem occur when i incubate with another antibody (after 2 rounds of stripping), it show the similar result as my first round? |
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By fkashani
on 23-Jul-2008
I have some problems with western please help me. |
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By Jai
on 17-Jul-2008
Hi, |
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By Sridhar
on 26-Dec-2007
better u digest ur actin ab to tht protein, |
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By Sridhar
on 26-Dec-2007
May be ur substrae concentration is more ,and also incubate your membrane long time with substrate. |
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By Dionne
on 25-Dec-2007
I am doing Co-IP, using high salt buffer to extract nuclear proteins. When running the samples in western blot, the input, which i took directly from the nuclear extract, could not be detected, but was detected in pull down. |
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By gb
on 15-Dec-2007
I got white bands in the western, standard has developed with low intensity. background comes very dark. |
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By Rajesh
on 15-Sep-2007
I have a problem with my western blots. The bands appear at the desired region but are not uniform. I don't think it is related to the loading. |
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By Lisa
on 20-Aug-2007
After I developed my western blot with ECL, I stored in in the fridge. After several days it turned orange. Why is this? |
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By shradha
on 08-Aug-2007
probably you may have forgotten to add 0.1%Tween-20 in your TBS-T buffer . This may be causing after ECL,black and spots darker than the rest of the film in your blot. |
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By Guadalupe
on 24-May-2007
Dear visitors, |
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By Johannes
on 23-May-2007
Dear people |
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By mitolab
on 25-Sep-2006
Few possible reasons for white bands and high background in western blot: 1. Too much protein loaded 2. Too high concentration of either primary or secondary antibody. |
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By xiaomei wang
on 22-Sep-2006
Dear Sir/Madam, |
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By J-science
on 19-Jul-2006
Dear visitors |
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By xumo
on 01-Jul-2005
good website |
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By elham
on 26-Jun-2005
Dear Sir/Madam, |
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