I did an SDS-Page and afterwards a Western Blotting to detect IgY form egg yolk of chicken eggs. One sample was boiled and the other was not. In my SDS-Page, there were for the boiled sample bands visible at heights of 25/27kDa, corresponding to the light chain of the antibody and bands at 70kDa corresponding to the heavy chain of the antibody. The bands of the light chain weren't visible anymore on the membrane, while the heavy chain bands were visible. What could be possible reasons for this?
Thanks in advance for the help!
My guess is that the antibody you are using in the western blot is raised against the heavy chain. Antibody probably has no idea that the light chain exists because it is probably not primarily binding to much of it. You can Coomassie stain the membrane to see if this is likely. If the bands of the light chain aren't visible with a simple Coomassie staining after electrophoretic transfer to a membrane and then staining the membrane with just Coomassie then there is a transfer issue. Adjust the electrical conditions of transfer or simply increase the time of transfer.
Charlotte van Langenhove
Use a 0.2 µm PVDF or nitrocellulose membrane. Optimize transfer conditions (time and voltage). Ensure thorough pre-wetting of the membrane with methanol. Equilibrate the gel in transfer buffer to remove excess SDS. Increase antibody concentration or incubation time. Include protease inhibitors and handle samples on ice. Optimize blocking and washing conditions. I hope you will get your desire band. Thanks.
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