My cell lysates are so dilute that to load 20ug of protein into my gel, the sample volume is too big for the wells.
I have heard of using MWCO filters, and I have had suggestions to let the cell lysate air-dry and reconstitute in a smaller volume of RIPA buffer + protease inhibitors. Are there any pros/cons to particular methods?
Any protocols would be greatly appreciated!
Hello Rebecca Miller
You may use SpeedVac to concentrate the cell lysates. This process takes place under vacuum conditions, which promotes solvent evaporation in the speedvac chamber. Samples are maintained in liquid state at sub-ambient temperature throughout the concentration process, preventing loss of activity or damage to heat sensitive substances.
You may spin the sample until enough solvent has evaporated and you feel that you have achieved the desired volume for loading in Western Blot. You may do a protein estimation of your cell lysates before you use them for Western Blotting.
Best.
Both methods are efficient, MWCO filters also led you to make buffer exchange additionally. Freeze-drying may lead to concentrating small molecules also along with your proteins and this may be a bit problematic for your PAGE (streaking, background issues, etc...) analysis especially if your protein is at a trace amount. You may try TCA precipitation and subsequent cold acetone cleanup. This will both enrich and wash your protein, thus your proteome including your target would be concentrated but also small molecule free...Indeed this suggestion should be verified by an experimental approach and by performing BCA/Bradford assay to see fold changes.
TCA precipitation is the way to go. I had that problem with a protein expressed in HEK293 and TCA precipitation helped. I works amazing, there are some small diffrences in protocols but all of them consist in precipitate with TCA 10%v/v in ice bath or cold for 20-30 mins, then washed with cetone 80% in TrisCl.
Thanks everyone! I'll give precipitation a go as we don't have a SpeedVac in our lab. Will let you know how it turns out :)
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