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What is Pellet that forms AFTER quick spin of boiled lysates?

extraction lysate sds boil pellet

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#1 chrizon

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Posted 02 August 2012 - 03:56 PM

I'm trying extract and denature proteins for a crude whole cell lysate adding buffer directly to cells. Everything seems to be fine until I short spin the lysates after boiling and notice a gel-like pellet has appeared.

Cells:
Primary Human Fibroblasts

Buffer:
5% SDS
60mM Tris, pH 7
10% glycerol
5% BME

Workflow:
-Add buffer directly to washed cells.
-Scrape/collect into centrifuge tube.
-Add nuclease (final amounts = 5mg/mL MgCl2, 100ug/mL DNase, 50ug/mL RNase)
-Let stand for ~10 minutes or until lysate could be pipetted easily.
-Spin @ 21600g for 10min, 4deg.
-No pellet at this point, but transferred about 3/4ths of lysate to new tube in case any nucleotides were still present. I assume that I ended up transferring too much because, in 2 of 4 samples, a viscous fraction broke loose near the bottom of the tube and ended up getting transferred anyway, so I added more nuclease. Samples became cloudy--this happened when I originally added nuclease to the samples, but it eventually went clear. This time it did not.
-Boiled samples 10min 95deg.
-Samples cleared up quickly during boil (within 1min). No observable precipitates.
-Quick spin up to ~10000g, then let go of the "short" button.
-Tada!! Pellet!
-Transferred equal volume (~90%) of each sample to new tube, conducted protein assay (660nm Protein assay, Pierce).
-Froze -80deg.

I tried resuspending the pellets in the remaining tube volume (~10% of original), but they weren't budging. So I fished it out with a pipette tip and it was a solid goo that could be rolled between my fingers without breaking up. Probably the most intriguing part is that the cells of 2 samples were treated with a reagent that is a transparent yellow in color, and these pellets came out orange. The other 2 pellets were opaque white.

Any ideas what I'm spinning down?

#2 bob1

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Posted 02 August 2012 - 06:40 PM

The clear viscous stuff is DNA in your original lysis. The DNase probably won't work in the presence of SDS!

You should only need to boil for 5 min to denature, any longer and proteins tend to aggregate (similar to cooking an egg - if you don't cook it for very long, the white stays clear, when you cook it longer the white goes white with aggregated protein)... you pellet is aggregated protein and is usually very insoluble. You could try adding a range of solubilizers such as guanidine and urea, but I don't think you will have much success.





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