Technique / Proteomics and protein biochemistry / Western blot assay

Western Blot troubleshooting

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	By fkashani on 23-Jul-2008 I have some problems with western please help me. I have two non-specific bands and no specific band, and this problem is detected with 3 diffrent "first antibodies" in the same patern, but in one of them the specific band is also detected. Rating: Good Reply

	By Jai on 17-Jul-2008 Hi, I have done the western blot for 2 proteins that contains the same antibody.western blot results showed positive for 1 protein but there are lots of non specific bands for other protein?can anybody guide me what could be the reason?Your feed back will be much appreciated. thanks Rating: Good Reply

	By Sridhar on 26-Dec-2007 better u digest ur actin ab to tht protein, then u'll come to conclusion if ur loading is not good.Mainly because of tht loading only u got the diference like tht. all the best Rating: Good Reply

	By Sridhar on 26-Dec-2007 May be ur substrae concentration is more ,and also incubate your membrane long time with substrate. the minimum time required for substrate digesion is 15min,if u digest more than this it'll develop dark background with non specific binding. Rating: Good Reply

	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. Can any one advise me? Rating: n/a Reply

	By gb on 15-Dec-2007 I got white bands in the western, standard has developed with low intensity. background comes very dark. what could be the possible reason! Rating: Good Reply

	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. Lack of uniformity of the band interferes with the quality required for a journal. Please advice. Rating: Excellent! Reply

	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? Thanks! Lisa Rating: n/a Reply

	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. Rating: Good Reply

	By Guadalupe on 24-May-2007 Dear visitors, After ECL, my blot came out all black and with spots darker than the rest of the film, but these where not the signal i was looking for. Where did I do wrong and what can I do with this blot? Thanks Rating: Good Reply

	By Johannes on 23-May-2007 Dear people i want to quantify my westernblot. In my case i want pool a couple of blots so do u know a way of finding a standart? Rating: Good Reply

	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. Rating: Very Good Reply

	By xiaomei wang on 22-Sep-2006 Dear Sir/Madam, After ECL, my blot came out like a nigative film ( where I should see the protein bands were clear, but the surounding area is gray). Where did I do wrong and what can I do with this blot? Thanks Rating: Excellent! Reply

	By J-science on 19-Jul-2006 Dear visitors i have a technical problem w/ the western the blocking part was suppose to be left overnight in a fridge...but instead i left it on an incubating rocker for overnight how badly did i mess up? how can i clean up this? thanks Jay ps. great website, thanks Rating: Excellent! Reply

	By xumo on 01-Jul-2005 good website Rating: Good Reply

	By elham on 26-Jun-2005 Dear Sir/Madam, I have some problems with western while using PVDF membranes after 2DE, althought I've done all the steps by protocols ( Amersham) but I did not observe any spots. Would you please guid me. Thank you in advance. Best Regards, Elham Erami Rating: Fair Reply