I keep trying Western Blotting using lysozyme antibody from SC and abcam, however I couldn't detect the band. Is there any suggestion that I can get through this?
I used donkey anti-goat for a secondary antibody as recommended in the data sheet. I also used a method of HRP staining and tried to detect the band using LAS.
First, you will need a positive control. Some Abs may detect overexpressed but not endogenous proteins. Or work in IF and not in WB. Then you can try to use a more sensitive assay than the for developing the peroxidase, althugh this may required some obtimization.https://tools.thermofisher.com/content/sfs/brochures/TR0032-Substrates-Western.pdf
based upon the information that you have provided, it is very difficult to determine a cause for lack of bands. I agree with the post above in that a positive control would be very helpful to determine the efficacy of the antibody. General questions:
1. How much protein are you loading
2. Do you homogenates have protease inhibitors
3. Have you tried ponceau staining to determine if the transfer was successful
Thanks to Victor and Brandon for great comments!
Sum up the comments, i need a positive control for Lysozyme Ab.
The amount of protein was enough to detect the band, and also using pi. Yes, i have done the ponceau after transfer also..
hmmm let me first try with the positive control
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