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Slimy protein lysates - (Jan/26/2009 )

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!

-Jay Em-

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.

-rkay447-

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?

-Jay Em-

Jay Em on Jan 27 2009, 11:15 AM said:

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

-wmw-