Protein denaturation without Laemmli sample buffer

Go to the profile of disaster_scientist
Mar 07, 2017
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heyy

during the sample preparation for western blot analysis, I made a silly mistake:
i heated my samples up for 5 min at 100°C without any loading buffer (2x laemmli) - just the pure sample.

Can I still use it? What happen to the samples, if you boil it without any sample buffer?

all the best & thanks for your help
disaster_scientist

2 Comments

Go to the profile of Roberto Rosati
Roberto Rosati 8 months ago

The proteins might still have some intact disulfide bridges (Laemmli contains a reducing agent, beta-mercaptoetanol). They might also have precipitated. Apart of that, if the pH of your solution wasn't too harsh, my opinion is that they probably didn't degrade.
All in all, if it's an irreplaceable sample or if you just want to know what happens, I would add Laemmli and heat again, according to the protocol. Worst case scenario, if you see a smear you know that your protein got degraded and you'll have to redo the experiment.

Go to the profile of Roberto Rosati
Roberto Rosati 8 months ago

P.S.: make a note of it in your lab book anyways. Even if it didn't affect the experiment, it doesn't mean it didn't happen.