I recently did a protein extraction from differenciated 3T3-L1 cells .
The fat form a layer at the top and cell debris form a pellet. At this point,I tried to remove the fat by pipetting or go under the fat to pull off the clear lysate with no success (Lysate still turbid)
Do you have any tip to get rid of this layer?
thank you in advance
What do you want to use the protein lysate for? Are you going to use it for western blot or IP experiment, etc? If you are going to use it for western blot I use a lysis buffer that is 8 M Urea and 1% SDS, adding 150 uL to the pellet. I incubate on ice for 10 minutes then ready to use. It is a very harsh lysate, but for some assays it is very effective
Hi Thinhinane, I would have two suggestions:
1) Collecting the liquid phase below the lipid layer is normally a good approach, the trick might be to use a very thin tip (for example the ones for gel loading) or a thin needle, gauge G27, staying away from the surface. Second trick is to repeat the centrifugation and collection of the solution below the fat layer at least twice. Also, slower centrifugation speed (10-12000 g) works best to compact the lipids on the surface.
2) Some people use the trick of the Qiagen RNeasy MinElute columns... just load your lysate and spin, you will collect as flow-through the protein extract without RNA and lipids... Just a bit expensive...
Good luck!
Thank you all for your input . Here some more details:
Shawn Krueger: I use it for Immunoprecipitation
Alessio Lepore : I used a TEM buffer : 75mM Tris pH:7.5 , 2mM EDTA, 12mM MgCl2, 2mM orthovanadate, 10mM NaF, PIC (Protease Inhibitor Cocktail)
we use an IP buffer like TEM buffer but with addition of triton x-100 to lyse the cell.
cell was collected by this IP buffer and sonicated in 1.5ml tube, then centrifuged by 13000 rpm at 4℃. Just like you said, there will be 3 parts, the top is fat, the middle layer of lysate and the bottom of cell debris. We usually collect the cell lysate using a needle or syringe for IP or co-IP experiments.
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