I have precipitated cell culture supernatants with TCA (10%) followed by overnight incubation and acetone washing...then I have added Lammeli sample buffer, but I am unable dissolve the protein still protein in the form of precipitate....
and with cultured cells, I have lysed them RIPA buffer and collected supernatant, and similarly I have precipitated it.
Both are becoming insoluble....how to proceed, I'Il need to go for a Western blot.
because they contain too much proteins now! culture supernatant medium must be containing FBS/FCS so they might be precipitated too! may be try adding the supernatant of this precipitate after spinning down.
Why you want to precipitate RIPA lysed cell lysate? you can directly use the cell lysate that are soluble,
Dear Elamurugan,
Sorry to say it, but your problem will be hard to resolve.. The tips about TCA I'm describing here:
https://www.researchgate.net/post/Can_you_show_the_picture_of_your_worst_Western_blot_and_explain_the_reasons_for_this_disaster_please/2
The other trick is: if you precypitate 100ul of cell culture supernatants (are protein rich) with TCA, dilute them 10x prior the TCA. This will give you a nice thin almost invisible pellet spread all around the eppendorf. It is much easier to wash of TCA out of the layer, than the fat pellet sitting on the bottom of the tube.
Best - Jan
if reduction of the disufide bonds is not important, you can use [sodium dodecyl sulfate (SDS): 2% w/v and dithiothreitol (DTT): 1.25% w/v] following a few minutes pipetige, it will redissolve most of the proteins.
Also you can use 10%TCA/acetone precipitation :
Chen, Y.Y., et al., A modified protein precipitation procedure for efficient removal of albumin from serum. Electrophoresis, 2005. 26(11): p. 2117-27
Avoid that the pellet dry during a long time, only the time required for acetone removal.
Try to sonicate the pellet or increase detergent concentration for improving proteins solubilization
Any success yet? I dissolved my pellet in the SDS-PAGE running buffer.
i could not able to dissolve the pellet...i am trying for reverse osmosis with sucrose to concentrate the protein...
spin your sample !GENTLY! down otherwise you transform your pellet into a neutron star-like object, you never-ever get dissolved anymore.
Hi.
I have a different problem while doing TCA precipitation with cell supernatant.
After centrifuging the supernatant with TCA, I can see pellets.
But I cannot see any pellet after washing with cold acetone (500 ul supernatant with 500 ul 10% TCA, then on ice for 30 min).
Or I can see smaller pellets after washing (1 ml supernatant with 400 ul 40% TCA, then in freezer overnight).
In the latter case, pellets are not white, but just transparent or hardly visible.
Anyway I proceeded until resuspending with 2X SDS sample buffer, plus 1-2 ul of 0.5 M Tris base.
But I couldn't have good results in Western blots.
What can be problems?
In addition,
when I used cell supernatants containing FBS, I could see big pellets.
They were still clearly visible after washing.
So most of them were actually FBS, I think.
These pellets were almost like powders that were hard to be dissolved after air dry.
After these samples were run, I found that the gel was messed with a crooked ladder).
Leave it overnight in the fridge in he following solution: 20 mM sodium phosphate, 0.5 M NaCl, 50 mM imidazole, 6 M guanidine hydrochloride, pH 7.4. If not dissolved completely, pipette gently up and down using a large pipette tip.
If the TCA pellet is intended for SDS gel analysis, then add SDS gel denaturing buffer (Tris-HCl, pH 6.8, 10% SDS, 0.6 M DTT, 30% glycerol and BPB dye) and boil for 10 min. The TCA pellet will dissolve for sure. However, if one want to dissolve the TCA pellet in a buffer intended for other use, then it will be difficult. One can try Rianita Van Onselen's suggestion: "Leave it overnight in the fridge in he following solution: 20 mM sodium phosphate, 0.5 M NaCl, 50 mM imidazole, 6 M guanidine hydrochloride, pH 7.4. If not dissolved completely, pipette gently up and down using a large pipette tip"
I had the same problem due to insufficient drying of acetone after TCA precipitation. Once your proteins are in SDS denaturing sample buffer, if you still have a pellet, alternate between heating your samples 95°C for 5 min and vortexing for 15-30 s. My pellet finally dissolved after 4 times.
my Problem is also a bit different.
i want an active protein after the precipitiation.
it is quite stable, but i am quite sure DTT will not help me.
also if there is a possibility without imidazol, i would appreciate or else i have to remove it again after disolving.
at the moment i use a buffer containing CHAPS, TritonX 100 and NaOH
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