Depending on your sample volume, I've had the best luck doing protein extraction from human brain using a Chloroform-Methanol-Water precipitation with 100-200ul starting material and 450ul MeOH, 150ul chloroform, 350ul H2O, and several MeOH wash steps with sonication and it can all be done in 1.5mL tubes.

The other alternative that has worked in my hands (for starting volumes >200ul) is to just acetone precipitate- add 8 volumes of -20C acetone to the sample, incubate 1-2hrs (or overnight) at -20C, then spin it down and resuspend the pellet in an appropriate volume.

I prefer the C-M-W method (splitting larger volumes up between several tubes for the first steps then combining the "protein pancakes" for washes) because even though there are a lot more steps, the final protein pellet doesn't seem to be as "sticky" and I feel like I don't lose as much from getting stuck to the sides of a tube, but I've never actually run the numbers to verify.

 A lot of people also use TCA precipitations, but I'm not sure what advantage that holds and we don't use the method in our lab.

Thanks a lot

Can I ask you how much tissue amount you had and how much protein yield?

Thanks, Ingrid

So I tired overnight aceton precipitation and cannot get the pellet dissolved in my buffer even after vortexing vigorously. Any advice for that. The buffer is as above.

Thank you,

Ingrid

for the CMW precipitation we aim to start with around 100-300ug protein in 100-200ul. but it works with as little as 50ug in ~200ul.

we've used the acetone precipitation with a subcellular fractionation method so I'm not sure the starting concentration... that's usually about a 0.5mL of sample to start and then after we precipitate and resuspend in 100ul the concentration ranges between 1-4ug/ul, but the acetone pellet is REALLY sticky and I'm sure that I lose a lot to the tube. We sometimes spike the resuspension with 1% of either/both Triton or SDS, and if we can't use detergent we'll sonicate to try and get it more homogenous.

I know the CMW method seems a lot longer, but how much easier the pellet is to work with at the end makes it completely worth it. 

Thank you Toni for your clear and honest reply. I will try this other option.

Most of the protocols based on precipitation causes protein losses. I would suggest lyophylization or dyalisis against solid sucrose. Alternatively you may use a precipitation protocol for extraction (i.e. TCA-acetone).

If you want measure protein activity, I'd recommend to avoid the organics. You can use those spin filters, they are available in several sizes with various cut-offs for your convenience.

You can use ammonium sulphate solution for concentrating proteins. This is called salting out, afterwhich you could then subject the precipitates to centrifugation. The salted out proteins would pellet for further recovery.

I would need the protein for cytokine analysis where it should be a very mild buffer they recommend, I am just relatively low with my protein concentration, so I wanted to concentrate it.

Thank you

if the protein concentration is low, ammonium sulphate precipitation is not very good technique.

centrifugal filters are on option. I use 30kDa cutoff for 46 kDa protein and 10 kDa for 16 kDa protein. I recomend those with the filter not on the base of the sample but along the sides. The dead volume might be larger but they clog less often.

Are you after specific types of proteins or do you want everything?

In regards to the pellet from TCA or CWM, resuspend it in Guanidine-HCl 6M ( I would recommend over Urea because you can heat it without carbamoylation on your proteins or other in vitro artifacts) . I can normally get rid of the pellet after adding TCEP  for reduction ( DTT can be used as well) and alylation using CAA (or IAA, IAM w.e) . Heating and shaking following reduction and alkylation normally dissipates the pellet in my experience. Pellet is normally not an issue especially once you add Lys-C to break it down it should all be digested. Remember to dilute down the Gnd-Cl to below 2M before adding Trypsin!

Hi

If your protein extract volume is low (1ml-15ml) then try to use 3KDa cut off centricon of millipore to concentrate protein samples. You can decide cut off value of centricon according to desired protein range.