Bettina,

Unfortunately I can't open up your image, but I will try to help. First, acetone precipitation is the easiest method. I assume your using TCA because it is a large protein sample.

TCA (80% w/v) is added to the protein sample to a final concentration of 4%. It is difficult to determine your final % from above. It is then incubated on ice for 30min, centrifuged at 15,000 x g, 4C for 10 min (missing from above), and then you decant the supernatant (missing from above). Add an equal volume of 80% cold acetone to wash away residual TCA (which can interfer with down stream assays), vortex vigorously, and then centrifuge at 15,000 x g, 4C for 10min, and repeat four times. Dry the pellet inverted on a paper towl for 30min, and then resuspend the pellet in SDS-sample buffer. This method is straight out of current protocols in protein science, and I've used it several times sucessfully. Also, make sure you're using acetone-compatible microtubes. Good luck!

V/r

Jonathan

Thank you Jonathan for the answer. I usually use our protocol and until now it worked fine.

I attached the picture again I hope you can open it now.

My first idea was that the protein amount is to high but as I wrote before we diluted already up to 1:20

My method is pretty similar to Jonathan's above, although I use a final volume of 10 % TCA.

A few other points:

- Can you see the white protein pellet after TCA precipitation/centrifugation? It should be pretty evident.

- 2 hours seems rather long for the TCA precipitation. I remember noticing some loss of protein concentration one time when I was lazy and precipitated overnight. Maybe some of your protein is degraded?

- What exactly is in your SDS-sample buffer? It has to contain something capable of re-dissolving the protein. I use 8M UREA in mine, and add to a shaking heat block at 50 degrees for an hour to help get the protein into solution. It can take a little while, especially if there is a lot of protein.

- How are you trying to detect your protein by Western - is it tagged, or are you using a native antibody? Is there also a positive control on the gel to ensure this step is working?

I can't see your gel file either btw

Is the gel homemade, or precast? My first guess would also be too much protein, but the streaking could also be due to contamination in your sample. Sometimes DNA in the sample can do this, but seems unlikely in this case. It might be due to TCA not being washed out properly

When you run the diluted and undiluted samples next to each other on the gel, do they look different?

Those are some great questions Rob.

Bettina,

I can see your gel now, and there appears to be some high MW protien bands. Esentially the lanes did not run properly, whereas your ladder did run properly. I'm assuming your ladder is a stock that may not be prepared in the same manner as the other samples were.

There are several things that may help. If you use loading buffer containing bromphenol blue and it turns yellow then there is still some acid left in your precipitate. You can neutralize this with 1M Tris of whatever pH you use for the stacking gel.

Also make sure you boil (95C for 10min) your samples just prior to loading them into the lanes to allow SDS to fully coat the peptides. Ensure that you use DTT or 2-mercaptoethanol in your sample buffer to help with denaturement. You can also 8M urea as Rob described above.

There are limitations to how much protein you can run on a gel. Perform BCA or similar assay to determine the total protein loaded. I certinaly agree to try without BSA, perhaps a Optim-MEM, which I have used sucessfully before.

Good Luck!

V/r

Jonathan

Did you add Tris base to neutralize contaminating TCA after resuspension in sample buffer? Your loading buffer should have bromophenol blue as a pH indicator. This may be obvious, and something you've already addressed, but if your sample appears green or yellow, it means TCA is still present. If the pH is off, your samples will not resolve well on the gel. From the looks of your gel, it appears that excess salts or pH problems are mucking up the proper migration of the bands.

- If the sample appears yellow, generally i add 1µL of concentrated NaOH and it works very well!!

- if its not a probleme of sample, may the water tah you use have a abnormal pH (i had a migration probleme and abnormal pH of water was the cause)

Hi Bettina,

Indeed, the gel looks weird. I agree with most suggestions above. I am not sure by yes you can omit BSA during your precipitation step. I have never tried to precipitate protein from the culture medium but 70% TCA is quite high. Normally it would be OK with 25-30% final concentration. Also wash a couple of times with HCl : Acetone (1:100 or even 1: 1000) to remove rests of TCA and then 3 times with acetone alone. I would first optimize the SDS-PAGE and then go for WB. Make sure you dissolve well the pellet. It is well known that it may get hard to dissolve after TCA precipitation. Also, remember some proteins may be soluble in TCA so they don't precipitate..

If it doesn't work then you can try an SPE cartridge (C18 or less ). You don't need to be expert in chromatography. Personally I would go for this. So, come back if you need to go this way or make a new thread if you need help.

There are a lot of good responses above. To add my own thoughts into the mix, I helped a colleague with precipitating proteins from the media of cells with 0.1% BSA. We had the advantage that it was a stably overexpressing cell line for our protein of interest but we also checked endogenous secreted proteins and it worked well. We found that higher amounts of TCA caused similar problems to what you are seeing - particularly if the acid is not neutralized as noted above. We did the following:

1. Take aliquot of media (we used 500ul) and add equal volume of 10% TCA - to give final volume of 5% TCA.

2. Sit on ice for 30 mins.

3. Spin for 5 min at 12000rpm to pellet the precipitant.

4. Remove supernatant and, to pellet, add 60ul of 2% SDS, Add 20ul of Tris pH7.4 and 20ul of loading buffer. If samples appears yellow, add additional Tris to neutralize acid.

5. Resuspend by vortex shaking for 5-10mins (you can tape tubes to a vortex if you dont have a vortex shaker)

6. Boil for 5-10mins and load an aliquot on a gel.

We had considerably more success with the lower % TCA precipitation than higher %, and didnt have major issues with BSA in the media.

I hope this is of use.

1. Even without TCA precipitation you'll see a thick smudge around 70 kDa and is due to the proteins in serum (FBS). You can see that the 70kDa region of last lane in right is pushed to right by the previous lane. If you can use serum free media (if your experiment design allows) this pushing effect can be avoided.

2. TCA precipitation leads to loss of most of the low molecular weight proteins (either due to TCA mediated degradation or during the acetone wash steps). If your target protein is less than 40 kDa or above 130 kDa (this is the MW range the serum proteins can smudge the banding pattern) just load 35ul conditioned media (without TCA precipitation)+8ul of 5x dye (boiled for 5 mins at 95-100 degree C) in a 1.5mm thick gel 10 well mini-protean comb.

3. As suggested above, if your sample turns yellow, add the stacking-gel-Tris a little, to neutralize pH until yellow color is lost. If the sample is yellow while loading, it will make the gel brittle.

4. If your target protein is low abundent, use immunoprecipitation to concentrate it. This can overcome all your problems.

5. If you know your target protein size, use appropriate MW cut-off columns to concentrate the conditioned media (a way to bypass TCA mediate problems).

Good luck..!! :)

Thank you all for your answers!

We do neutralize the the sample buffer with Tris-Base when turning yellow and usually the pellets solve completely. We cook at 95°C for 5 min.

I will try less TCA but still its kind of strange that the protocol used to work for years now. Even in those cells.

I will perform the experiment together with my bachelor student for she did it alone last times. Hope it will work!