HI all. I was preparing my samples for Western blot, and accidentally boiled my proteins at 100C for around 8 min instead of the intended 5 min. My proteins of interest did not show up on the membrane, but my GAPDH did quite nicely. Can I say for sure that my proteins did not degrade due to the excessive boiling, and that it's another issue?
I would think that it's another issue, since an extra 3 mins shouldn't make much of a difference. But there's the small possibility that GAPDH is of a higher quantity than your protein of interest and hence even if 10-20% of the GAPDH in your sample did degrade, you'd have a GAPDH signal from the remaining.
Hi Carey, if you were boiling your proteins in 1M NaOH or 1M HCl then proteins would degrade. In SDS sample buffer? Nah.
Some proteins, particularly membrane proteins, seem to disappear from SDS-PAGE samples when they are boiled. In those cases, it is preferable not to heat the samples.
With LDS sample buffers, the samples are heated at 70oC, not boiled.
If your protein normally shows up after 5 minutes of boiling, I don't think boiling for 8 minutes will make a difference.
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