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Problem with thawed samples


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#1 steinpdh

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Posted 29 September 2014 - 05:54 AM

A few months ago I have prepared several batches of samples (whole cell homogenates in Laemmli buffer) the exact same way every time. Every batch I would aliquot in volumes of 50 µl. One aliquot I used for an experiment right away, the others were frozen at -20 °C. They would all yield beautiful results on my gel.

 

Now, about 6 months later, after thawing, some of them work just as nice as the fresh samples, but some don't. Gels turn out completely ugly. They resemble samples with e.g. too much potassium in them, with lane narrowing and smear and all that. Though there's no potassium in them.

 

What else could be causing this? Is there anything I need to pay special attention to when thawing the samples?



#2 mdfenko

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Posted 06 October 2014 - 05:10 AM

other salts can cause what you're seeing but that is probably not your problem.

 

you're probably seeing the effects of protein aggregation. proteins can aggregate (even in the presence of sds) when frozen.

 

make sure that all of the sds is back in solution after defrosting (the most likely cause of your problem).

 

if the problem persists, reboil the sample (not too long, over-boiling in sds can cause aggregation).


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#3 steinpdh

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Posted 07 October 2014 - 06:50 AM

trying to follow your advice, after thawing the samples at room temp i have heated them twice for 10 minutes at 60 °C with a bit of vortexing and a short centrifugation in between and one final 5 min centrifugation right before loading. and it kind of worked! at least it looked much better than before, although not as good as some other samples (that i didn't even need to heat). i still had a few vertical streaks through bands of larger proteins and bands were generally still not as sharp as i would like them to be.

 

would you recommend heating/mixing even more?

 

one more thing: the samples were originally heated in Laemmli buffer with DTT, of which i thought it would irreversibly break the disulfide bonds, and they worked fine the first time. but could adding a bit of fresh mercaptoethanol to the thawed samples before heating still make sense, or is it definitely redundant?



#4 mdfenko

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Posted 07 October 2014 - 07:06 AM

vertical streaking above the band is caused by aggregates, slowly disaggregating. below the band is usually caused by decomposition.

 

reduction of disulfides is not permanent. bme and dtt will both oxidize over time and so will the sulfhydryls. adding additional reducing agent will help.

 

you can also increase incubation time at 60C (20 minutes is not too long).


talent does what it can
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i do what i get paid to do




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