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too much heating LDS-sample buffer

western blot lds-sample buffer

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3 replies to this topic

#1 murusa

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Posted 17 July 2014 - 01:49 AM

Hello all! I have one question about preparing samples for the western blot.. usually I use LDS-sample buffer with b-mercaptoethanol and heat samples at 70 degrees during 10 minutes. But last time when I finished heating I suddenly noticed that the temperature was higher, maybe about 95 degrees. the heating time was the same - 10 minutes..

I wonder what I will see on the western blot in this case? will that heating destroy my samples and I will see an empty wells after staining? I want to check IKK, p-IKK, JNK and p-JNK.

 

thank you in advance! 



#2 mdfenko

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Posted 17 July 2014 - 04:10 AM

spin the sample. if you get a significant pellet then the protein may have aggregated.

 

but, 10 minutes at 95C should be okay (maybe even better than 10 minutes at 70C)


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#3 murusa

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Posted 17 July 2014 - 05:37 PM

thank you for you reply, nice idea about spinning.. I will try!

but in case I get some pellet (aggregated protein) - it means that I cannot get a good gel electrophoresis because big aggregates cannot move properly right? so it will look like no expression at the stained membrane?

 

I know usually heating 95 degrees is good for SDS-sample buffer but in case using LDS-sample buffer they recommend heating only 70-80 degrees because LDS-sample buffer is sensitive to heating... that's why I asked how overheating could affect my result.. 



#4 mdfenko

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Posted 18 July 2014 - 10:43 AM

i can't find anything saying that the lds is particularly susceptible to heat. it's probably another component of the buffer that is sensitive (maybe the reducing agent) and they are all probably part of sds sample buffer. so the sample should probably be okay.


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