Whatever Qiagen suggests, acetone is not the best way to precipitate protein from complex mixtures, as it precipitates nucleotides, salt, and many other small molecules you don't want there. The best method of precipitation is 9 volumes of methanol, followed by wash with 90% methanol (e.g., as in Neurobiol Dis. 2011 Nov;44(2):248-58). Methanol precipitates protein even from very dilute samples (just let it stand ~10-15 min at room temp), and it does not precipitate salt or nucleotides. This pellet should then be dried (not over-dried, only until it stops smelling of methanol) and dissolved in SDS sample buffer, after which it is ready for electrophoresis with subsequent Western, etc.

From mild extraction and clean Western to harsh extraction and dirtier Western. It's a trade-off, and no one answer for each protein exists. Options: 1. Sonicate vigorously after adding RIPA buffer, leave at RT for half an hour or so, and vortex or sonicate again every now and then. The entire pellet won't dissolve, but thats ok. You basically extract the pellet which has other insoluble crap. Spin down and use Sup to load your gel (with loading buffer). This will give pretty clean Western results and your protein is probably in the sup.

2. Do the same but increase SDS to 1% (was 0.1) and NaCl to 400mM (was 150mM). This will extract/dissolve more.

3. Harshest; Add 1.5x protein loading/sample buffer directly to the pellet. Vortex or sonicate. Boil 5min (90-98C). Vortex/sonicate again. Spin down crap. Load sup.

If you still have trouble with the acetone pellets, try dissolving in 0.05M sodium hydroxide. When dissolved (or substantially dissolved), add 0.05M HCL to neutralize, then add 1/10 10X Tris, pH8 (or other acceptable buffer). This is even harsher than #3 above, but has been used to dissolve membrane proteins in plants. I think I might have a ref, if I can find it! Email me if you are interested and/or the other suggestions don't work well.

You should be able to just add your sample buffer and boil. Do you want to quantify your protein prior to loading your gel? then I would use a detergent buffer (triton X-100 or SDS). These will take time and some gentle heating and vortexing. After the pellet has dissolved, you can measure your concentration using BCA or some reagent not very sensitive to detergents in the buffer (Bradford won't work here). Good luck.

The only work you have to do is adding 50 ul of water, vortex carefully and then, 25 ul of Laemil 3X buffer - boil and run WB. I did it many times and suceedded.

You can dissolve it in Tris base ph=7.6 or 160mM tris pH=6.8 in addition to 2% SDS. In this case you need BCA for protein quantification.

Also, When acetone precipitating the protein, try not to leave the sample to dry out for too long.

Try to homogenize your pellet in 10M Urea/2x SDS sample buffer (1:1). Usually I use this one to solubilize tough animal tissues and dissolve almost everything. You don't need to boil.

I would addd that the Pierce 660nm assay can be used to quantify the amount of protein resuspended in Laemmli buffer (even with bromphenol blue added).

I would use 8M urea / 20mM DTT on the pellet after removing all the acetone via speedvac. Start with 50ul at first and work your way up depending upon the pellet's size. I'll usually put it at 37C and shake it until the pellet dissolves. Urea/DTT treated protein samples look GREAT on westerns. I'm doing two right now. Be careful not to overload your gels and protein quant it first. Good luck.

Also keep in mind that you extracted the total protein from the cell lysate, both soluble and insoluble. Not all of it will go into solution with just PBS etc. This is why Urea / DTT does an excellent job bringing in proteins like those sequestered in inclusion bodies and membrane bound proteins in solution.

Any aceton precipitate is very dehydrated. Therefore it is often useful not to increase the water contend suddenly. You could start with the smallest volume buffer possible and put it in a potter tube (which is a a glass tube with a very well fitting teflon stopper that allows you to disperse you material ovr a large surface without increasing your volume). If the protein does not have to stay native, boiling in sample buffer or Urea/DTT (freshly made) is a good option.

You can just resuspend it in loading dye (laemmli buffer).

You can wash the pellet with 70% acetone and 50% acetone and air dry till all acetone is evaporated. Then you can resuspend in any buffer. I have done that for pellets we routinely give for 2D-DIGE labeling and IEF and other assays.

Add the amount of desire loading buffer , boil up to 75 celcius degree for approximateley 15 min, even though you can not dissolve all Your proteins most of it will disolve in to the loading buffer.

I do basically what Jason Blum describes after acetone precipitation. Given the amount of protein, I usually add about 300 mL 5% SDS and heat it gently. I can assess protein concentration with Pierce assay and have a zillion aliquots left for Westerns.

I used to clean up samples for 2D gel-elfo with a similar method to acetone precipitation. Yes, it can be very difficult to dissolve the protein pellet. I used 2D lysis buffer (8M Urea, 2M Thiourea, 4% CHAPS, 50mM DTT) and depending on the pellet sometimes I also sonicated it. Of course, you need to peform protein quantification before loading the samples onto gel. Be careful with it because 8M Urea interfers with most of the assays. I used Non-Interfering protein assay kit.

Hi Alejandro!

I usually perform acetone precipitation to get my proteins in a volume easy to pipet and load in polyacrylamide gels or to clean up samples for 2D gels. As Istvan suggests, for 2D gels I use the 2D lysis buffer, for regular western blot I resuspend the pellet in 1X Laemmly sample buffer, denature and load into the gel. Resuspension it's easy and works every time for me.

Good luck!

I find that acetone pellets usually dissolve very quickly in just pure distilled water. Any solutes seem to interfere with rapid dissolution. I use a pipette tip to stir or, better still, a Pasteur pipette with the the end flame-sealed. It takes seconds for a small pellet in a microfuge tube (I'm not sure if the type of protein makes a difference, but I have done this with mammalian whole-cell extracts). Then just add 1/4 volume of 5x sample buffer.

I would add to dry pellet x1 sample buffer for the PAGE which has 2-4%SDS to dissolve your proteins.

I have done both - what Norbert and Irada (above) are suggesting. If I have more time I would add double distill water, let it stay for 5 -10 min at room temperature then vortex until I see the pellet dissolved, then mix with 2X Laemmli buffer. If I have less time I would add 1X Laemmli buffer and vortex, but it tends to make more foam. Once everything is in solution boil for 10 min and you are ready for loading. If you start boiling before the pellet dissolves and it stays as a pellet – try to break it with a tip (P200 works well). Sometime the pellet tends to stick to the tip. In this case I usually will put the tip in the Eppendorf tube, will put some water in the tip and will let it dissolve.

Try repeated extractions of the protein precipitate with 4 M guanidine thiocyanate.

Good luck!

Acetone precipitated Protein pellet is readily soluble in Urea/Thiourea buffer (7M urea/2M thiourea)

You can try to dissolve in 1% Triton-X-100. After that the dissolved portion should be recovered by spinning.

One important point I forgot to mention is don't let the pellet dry. Drying under vacuum especially makes it hard to redissolve. I remove all excess liquid by suction with a fine gauge needle, so only the pellet remains, then add water immediately to dissolve, stirring with a thin, drawn-out glass rod or equivalent (pipet tip is not great, as was pointed out), then adding the SDS buffer. Vortexing is not a good idea. Also, don't forget that there are still active proteases in the dissolved pellet. The shorter the time between adding the water and adding the SDS, the less protein degradation you will have. Urea works well, but you can't then subsequently heat your protein before gel loading because it will carbamylate your proteins and this may be a problem for further characterization, including antibody staining. http://www.ionsource.com/Card/carbam/mono0005.htm

Whatever Qiagen suggests, acetone is not the best way to precipitate protein from complex mixtures, as it precipitates nucleotides, salt, and many other small molecules you don't want there. The best method of precipitation is 9 volumes of methanol, followed by wash with 90% methanol (e.g., as in Neurobiol Dis. 2011 Nov;44(2):248-58). Methanol precipitates protein even from very dilute samples (just let it stand ~10-15 min at room temp), and it does not precipitate salt or nucleotides. This pellet should then be dried (not over-dried, only until it stops smelling of methanol) and dissolved in SDS sample buffer, after which it is ready for electrophoresis with subsequent Western, etc.

The suggestion of Vsevolod to use methanol is good, but the most reliable procedure for this in my hands is that of Wessel & Flügge:

@ARTICLE{Wes-84,

author = {D. Wessel and U.I. Flügge},

title = {A Method for the quantitative recovery of protein in the presence of detergents and lipids},

journal = {Anal. Biochem.},

volume = {138},

year = {1984},

pages = {141-143},

doi = {10.1016/0003-2697(84)90782-6},

language = {engl},

}

The protein is concentrated in a tight film at the interface between the water/methanol and the chloroform phase, after washing with methanol you get an easy to spot pellet even with very small amounts of protein.

The pellet ususally disolves well in SDS-sample buffer, but Mykhaylo's idea of using urea may help in difficult cases.

First of all ensure that all acetone has been removed. You may do this by a dialysis method. Now depending on the type of protein, find out its isoelectric pH (approximately if not accurately). Design a buffer accordingly where it can be dissolved.

I normally air dry the pellet (about 10 min) and then dissolve the pellet with 1x sample buffer (amount depends on the size of the pellet). It works fine with me.

Don't let the pellet dry - it's almost impossible to resuspend if you do. I used the following buffer to resuspend my precipitated plant proteins: 1% SDS, 10% glycerol, 10mM Tris-HCl (pH 6.8), 2mM EDTA, 160mM dithiothreitol (DTT). Add the required amount of buffer to the pellet in a 1.7mL eppendorf (depends how much protein you have and how concentrated you want it). Place this in some form of agitator such as a shaker or rocker on low at 4'C and leave overnight. In the morning your proteins should have dissolved - what hasn't dissolved is probably not protein.

but if i need to dissolve it to run on a native gel, so which buffer is the best without using any SDS?

Try to dissolve it in 2%SDS, and 60mM Tris pH=6.8

Anybody tried the method of using 9x volume methanol to precipitate protein that mentioned above? I tried to use this method to precipitate BSA from 1mg/ml water solution, but after I add room temperature method into the BSA solution and wait for 20 min, spin tubes at 14000xg, I get no pellet at all.

Try PRMM method, doi: 10.1128/AEM.70.1.610-612.2004

Haven't done an acetone precip. in over a decade, but I seem to recall that barely-dried pellets dissolve in a minimal amount of just plain distilled water. Use a flame-sealed Pasteur pipet as a stirrer to suspend/dissolve the pellet. Any ionic strength changes solution time from minutes to hours. You can add buffers etc. after dissolution. BTW, if you use urea, do NOT heat your sample above 37 deg. Urea and heat will transcarbamylate your protein (particularly messy for 2D gels). Methanol precipitation has to be cold, not room temp. if you want anything approaching quantitative recovery  for dilute mixed proteins.

i tried to use ammonium sulphate with which i did not get good protein recovery after the precipitation and dialysis. so i tried using 1: 9 volume of sample and ethanol. i get good precipitates but they do not dissolve completely in  sodium phosphate buffer although i get good recovery.....i still have to find out if the protein works well for characterization of temperature and ph and native page and sds page.

please let me know if anybody has any suggestions on how to complete dissolve the pellet and if this method is good enough

....................

http://www.monzir-pal.net/Bioseparation/Lectures/L9.pdf

The above link gives an excellent description of which buffer will work to re-solubilize proteins and how each method of protein precipitation works.  I can tell you from experience that RIPA buffer does not work that well on methanol precipitated proteins.

Best of Luck.

Michelle

Hi

I don't think the above link is active currently; could you please post a new one? Also, any recommendations on a suitable buffer for re-solubilization of proteins following acetone precipitation, without SDS or any harsh detergents?

Thank you!

Hi Iswariya,

Yes, it seems the above link is no longer viable.  Acetone precipitates proteins by "dehydrating" the protein and which causes the proteins to aggregate.  Of note, is the temperature of miscible solvents, which has to be below 0 degrees  to prevent protein denaturation.  Often a small amount of protein denaturation occurs during acetone precipitation. Samples precipitated using this method are generally used for downstream applications in which the  buffers used in re-solubilizing the sample, is needed for the downstream application, i.e. SDS containing buffers such SDS-PAGE sample buffers.

My only other thought would be to sonicate the samples if you do not want to add any detergents.  There are also many native gel sample buffer recipes that may be of some use to you.

Sorry I couldn't help.

Michelle

Hi Carole Leavel Bassett,

Your suggestion really worked about dissolving the protein pellet

The protein has been isolated from raw cell lysate.

Ma'am could you please provide the reference this?

I have found that the best way to resuspend a methanol/acetone precipitated plant protein pellet is to use the Sigma Plant Total Protein Extraction Kit PE0230- there is a reagent PER4 that does a terrific job getting the protein into solution and ready for Western blotting.