I want to concentrate my proteins using TCA, and do 1-D SDS PAGE. I have a few concenrns.
i) Will the TCA remove salts or concentrate them as well
ii) For resoulubilizing precipitated proteins, is the SDS loading buffer the only thing i could use. And if i use it then what should be the volume. Is there any other SDS containing buffer to resolubilize proteins
iii) how will i quantify protein concentration to be bale to load equal amounts in each well
iv) Would any other precipitation method , (e.g Acetone) be a better option
Kindly let me know. And if possible provide references for proper protocol
Thank you so much :)
Hi Pritish
Yes you can use 16% TCA to preciptate your proteins (15 min at full speed in an eppendorf centrifuge at 4°C is generally enough). It won't precipitate the salts (it can be used to get rid of salt that would perturb the migration).
In some instances (when you precipitate too much protein at a time) you can have trouble to get rid of the TCA that is caught within the pellet, so that when you redissolve your protein, your sample buffer will turn yellow because of the acidity of TCA (if it contains bromophenol blue) because of the acidity of TCA (even after the acetone wash). Then you should add 10 mM NaOH µl by µl to neutralize the acid until the samples turns blue. Also drive your sample buffer all over the pellet several times to redissolve it.
Also, I have observed that once precipitated by TCA, proteins have a tendency to degrade, so load your gel on the day you do the precipitation, and do not reuse samples once they are precipitated (aliquot before precipitation if you think you will need to load the same sample several times)
I am not sure I understand your question about protein concentration. Either you can normalize your samples from the beginning of the experiment (i. e. load quantities of extracts corresponding to the same amount of cells) or you measure the protein content by Bradford assay, the method of Lowry, or similar methods. If your concern is that the efficiency of the precipitation might be irreproducible, you can just add the same amount of a purified protein like BSA in all your precipitation. Anyway, the TCA pellet sticks quite well to the tube, even if it is not visible, so if you work carefully the precipitation should be reproducible.
TCA -precipitated proteins should be washed twice with cold acetone to remove TCA. 2-fold SDS loading buffer is usually used to solublize the proteins.
One important thing is that when you want to precipitate target proteins secreted from cells, you should use low levels of FBS or Opti-MEM without serum. Otherwise, the serum protein may overide your target protein.
For precipitated proteins, quantification may be unnecessary as different treatment may result in differing protein release. So use the same supernantant from each well with similar cells planted.
Hope having help to you
Correct, I´d only only add that it is 90% cold acetone (-20 degrees) and wash three times at least. If the gel runs odd, the wash more. Good luck.
Hi Pritish
Yes you can use 16% TCA to preciptate your proteins (15 min at full speed in an eppendorf centrifuge at 4°C is generally enough). It won't precipitate the salts (it can be used to get rid of salt that would perturb the migration).
In some instances (when you precipitate too much protein at a time) you can have trouble to get rid of the TCA that is caught within the pellet, so that when you redissolve your protein, your sample buffer will turn yellow because of the acidity of TCA (if it contains bromophenol blue) because of the acidity of TCA (even after the acetone wash). Then you should add 10 mM NaOH µl by µl to neutralize the acid until the samples turns blue. Also drive your sample buffer all over the pellet several times to redissolve it.
Also, I have observed that once precipitated by TCA, proteins have a tendency to degrade, so load your gel on the day you do the precipitation, and do not reuse samples once they are precipitated (aliquot before precipitation if you think you will need to load the same sample several times)
I am not sure I understand your question about protein concentration. Either you can normalize your samples from the beginning of the experiment (i. e. load quantities of extracts corresponding to the same amount of cells) or you measure the protein content by Bradford assay, the method of Lowry, or similar methods. If your concern is that the efficiency of the precipitation might be irreproducible, you can just add the same amount of a purified protein like BSA in all your precipitation. Anyway, the TCA pellet sticks quite well to the tube, even if it is not visible, so if you work carefully the precipitation should be reproducible.
Hi pritish,
I have also used TCA precipitation method to remove salt impurities, in my case GnHCl. So the answer to ur forst query is, it does remove salts and concentrate the protein as well.
After precipitaion, the protein pellet formed will sometime contains the TCA, which make the sample acidic. So when you mix your sample with sample/loading buffer it will turn yellowish due to high acidic content. You need to remove the traped TCA from the sample at the first place. This could be done by washinf the protein pellet with cold Acetone( at least twice). I have attached the protocol for TCA precipitationa nd preparation of protein samples.
Protein quantification can be easily done by either bradford or Lowry's assay. Also if the extinction coefficient of the protein is known, you can quantify using UV spectroscopy following the beer-lambert's equation.
One more thing, you can also increase the strength of loading buffer. instead of using 2X, you can go for a 6X loading buffer( in case you are getting a low concentration of protein in the sample). This will help you to load more protein as compared with 2X.
Hope this would be helpful to you.
Hello,
I want to remove SDS and DTT from the extracted protein as these additives interfere in the quantification by Bradford's method (Extraction Buffer: 3% SDS, 0.1% DTT, 0.125 M Tris-HCl, pH 7.5. I need high concentration of SDS in the extraction buffer).
Is the protein precipitation method described by Kanti will be helpful in removing SDS and DTT?
After quantification by Bradford can I suspend the protein directly in loading buffer.
What is the significance of increasing the strength of loading buffer to 6X. I remember, once I used 5X loading buffer and it distorted the wells.
Is there any other method that you can recommend (by your experience) to quantify protein that works well with the above-mentioned contaminants.
Many thanks in advance!
Nidhi
Hello,
I did the TCA-acetone wash precipitation for secreted proteins. My problem was low protein concentration and low precipitation yield to even quantify them. If you have a lot of proteins, go for TCA precipitation it's very cost effective! But with my low concentration, I ended up buying the amicon concentration filters and it worked perfectly!
For quantification, all papers I read do BCA of Bradford assay. I did BCA.
Use 2x SDS Sample buffer (100mM Tris-HCl, pH 6.8; 200 mM DTT, 4% SDS, 20% Glycerol and 0.2% Bromophenol Blue in distilled water) and add PBS to make it 1x in a tube with TCA-precipitated protein. Bromophenol blue in your SDS Sample buffer will act as a pH indicator. It will turn yellow below pH 4 and remain blue above that pH. Therefore, if the SDS sample buffer turns yellow, the solution is still slightly acidic due to remaining TCA. Just add a small amount of 1 M Tris pH 8.0, until the color returns to the usual blue. DO NOT use NaOH because you could hydrolyze the protein - hence, all the problems, especially when you have little starting material.
The 2D quant kit (GE Healthcare), provides specific reagents for protein precipitation prior quantification. It is compatible with SDS (2%) and DTT (1%). The precipitation is way much faster than the typical TCA/acetone method.
I am trying to isolate total protein from the ascomycete fungus Rosellinia necatrix. The problem I am facing is very low yield and when I boil crushed mycelia directly with 2X sample buffer and then centrifuge to get the supernatant, I get a mucilaginous jelly-like substance that makes it difficult to get the sup. Another problem I face is after TCA precipitation, when I try to boil that precipitate with 2X sample buffer, the pellet doesn't dissolve completely. Majority portion remains undissolved even after boiling. Could you please suggest me any solution? Thank you in advance!
Hi Subha,
Your problem might be DNA (or maybe polysaccharides, I don't know anything about your fungus). A thing that you can try is recover your sample in a sufficently large volume of sample buffer (200 ul) so that you are able to sonicate it (with a probe that you dip directly in your sample, not in a bath). It's a little tricky because you don't want to make foam so your probe has to be really close to the bottom of the tube, but in my hands 200 ul in a 1.5 mL eppendorf (important because the shape of the tube matters) for 10 s at low power works (overheating is not a problem since you boil the sample afterwards). I use it to get clean gels of bacterial crude extracts and avoid smears produced by DNA. I have never observed any deleterious effect of the sonication on the integrity of the proteins as seen on an SDS-PAGE.
Hope it helps
Olivier
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