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Slimy protein lysates


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3 replies to this topic

#1 Jay Em

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Posted 26 January 2009 - 06:20 AM

Hello,
after using this protocol for the extraction of nuclear and cytosolic proteins I sometimes end up with very slimy lysates after heating them up (95C for 5') before putting them on a gel. This is really putting me off..since I've tried amost everything to avoid this. i.e. avoiding to high protein concentrations in the lysates, spinning them down @ 13.000 rpm for 15 minutes before protein quantification and before loading the gel to avoid transfer of sediments...I'm running out of ideas...
Advice would be highly appreciated!

#2 rkay447

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Posted 26 January 2009 - 01:32 PM

Your nuclear sample should be very thick and difficult to pipette because of all the chromatin. Not sure why your cytosolic would be a problem unless you have contaminating DNA. Try sonicating the samples after boiling in SB to shear the DNA. Sonicate 3X at 10-15 seconds and then spin at 13,000 rpm for at least 5 mins. If your sample is still too thick you may want to dilute the sample further and try loading the sample onto the gel directly after boiling. Generally the hot samples are a little easier to load.

#3 Jay Em

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Posted 27 January 2009 - 02:15 AM

Thank you! You're right! It's mostly the nuclear lysates that are causing the problem... but what can I do?? Use DNAse? What can I do to make samples easier to load after boiling? Maybe spin them down longer and will the chromatin be in the pellet after that?

#4 wmw

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Posted 19 February 2009 - 01:45 PM

Thank you! You're right! It's mostly the nuclear lysates that are causing the problem... but what can I do?? Use DNAse? What can I do to make samples easier to load after boiling? Maybe spin them down longer and will the chromatin be in the pellet after that?


you can try using a syrynge and needle, pull sample up and down a couple of times and then try again.... hope this helps




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