I prepared my samples for western blotting and I did overheat my samples mistakenly after adding sample buffer (SDS) for about 20 min, now i did western blotting but got no clear result. Can anyone help to know it is too much to heat my samples and what happen to my samples if i overheat them!
Thank you so much.
Can you please send a picture of the blot? I've forgotten about my samples while heating them before and it didn't affect the outcome.
Many complex transmembrane proteins (like NaK ATPase) will completely disappear from SDS-PAGE and subsequent Western blots if heated to boiling, I found ~60-80C worked much better. Other proteins take boiling in SDS sample buffer quite well and its regularly used for SDS-PAGE gels and western blots. Overheating during western transfer can even lead to boiling and bubbles in the transfer in my experience; its best to keep temp low to avoid this. As stated a picture will aid in troubleshooting.
Every scientist at least once in their life have forgotten the sample on the heat block, that just happen! After boiling samples you should always spin it down and load the surnatant on the gel, that should solve you problem. When boiling for too long one common issue will be that the sample dries up, so putting your samples on the centrifuge will help all the evaporated water to get back in the sample. SDS is not affecting the results as it is in the gel and in the loading buffer. Good luck!
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