Strip Western Blotting Troubleshooting - (Jun/26/2012 )
I want to do a western blot on strips (of 3mm×~6.5cm).I tried the method like semi dry transfer and then cut the membrane (after blocking and storage at -200c ) and then treated with primary antibody(patients serum1:10, 1:100) , secondary antibody (anti human IgE-HRP) 1:2000 and DAB(also had some problems with DAB <0.03% H2O2 with mg /ml DAB> but the substrate was giving immediate colour formation ,so i tired to do this very fast immediately before adding ) .But the colour formation was not specific for the proteins in the strip (kind of background colour formation) .
Then I tried with Pierce ECL plus western blotting substrate and with secondary antibody dilution 1:10000 unfortunately i did not get any reaction for my proteins.Could anybody help me to solve this?
Thanks in abvance.
The first thing you need to do is trouble-shoot the process - see if there is protein on the membrane (ponceau S stain), if there is proceed to the western trouble-shooting. If not, you need to optimise the transfer process, which will depend on the protein (size, charge) you are looking at and the conditions you are using - try varying the methanol content and SDS content of the transfer buffer.
For the western - you probably need to titrate the serum concentration, you may need to go as low as 1:10 to get a signal. I would also recommend using the whole blot to do this - you may be missing the protein of interest by cutting the membrane too small.
Thank you for the reply.Today i tried with 4 chloronaphthol as substrate (just in a 0.5ml tube and a dot blot ) and it is giving me right result(only with my antibody).But it needs more antibody concentration because with 1:2000,1:10,000 no results but when i simply add 1microliter antibody it gave me strong colour(Abcam: antihuman IgE-HRP).
But i cant use the antibody so lavishly like that. The serum i am already trying with 1:10 dilution .Why i am trying with the strip is because i have only a small volume of patient serum so thought that once i got confirmed with the strips i can proceed to western blot.And I am cutting the membrane vertically so that i can get all the protein bands.Hmm anyway as you said i have to reanalyze each micro steps to find a solution.