Slimy protein lysates - (Jan/26/2009 )
after using this protocol for the extraction of nuclear and cytosolic proteins I sometimes end up with very slimy lysates after heating them up (95°C 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!
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.
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 on Jan 27 2009, 11:15 AM said:
you can try using a syrynge and needle, pull sample up and down a couple of times and then try again.... hope this helps