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Problems with precipitation of proteins using TCA/aceton - (Jan/25/2011 )

Hi all!
Im a student from germany!


Im trying to precipitate whole cell protein to get a higher concentration of my proteins for WB.

I use RIPA-buffer for cell lysis:

150 mM NaCl
50 mM Tris (pH 7,5)
0,1% SDS
1% NP-40
0,5% sodium deoxycholate
Protease inhibitor cocktail tablet
2 mM EDTA
1 g/ml Pepstatin
50 mM sodium fluoride (NaF)
1 mM sodium orthovanadate (NaV)
1 mM p-nitrophenyl phosphate (PNP)
1 mM Na--glycerophosphate


After 10 mins of lysis I add an equal amount of 20% TCA (4C) to the solution, vortex and let stand at 4C over night.

Then I follow this protocoll:


1.) Centrifuge the sample(s) at 15.000xg 10 minutes at 4C.
2.) Discard the supernatant properly.
3.) To the pellet add 800 l of -20 C acetone. Vortex the sample back to complete resuspension.
4.) Vortex and let sit at room temp for 5 minutes.
5.) Centrifuge at 15.000xg, 4 C for 10 minutes.
6.) Discard the supernatant.
7.) Repeat Step 5-7
8.) Discard the acetone and dry the pellet completely for 30 minutes at room temperature.
9.) Resuspend samples in desired buffer.
10.) Let the sample sit for over 1 hour on ice to solubilize all the proteins.




My problems are:


1.) I have a really big pellet after the first step but after the first washing step, it seems smaller. Is this a sign of protein loss?
2.) I cant resuspend my pellet in aceton for washing! The whole pellet is flying around. After mins of vortexing it falls apart, but in only ~10 pieces. Did I spin to fast/long?
3.) I cant resuspend my pellet after drying! I tried different times from 5-30 min drying (room temperature or fume hood), so I exclude the possibility of overdrying the pellet. I tried to resuspend in the RIPA-buffer for quantification. Why does my pellet not resuspend?

I will be very thankful for any help!

Greetings!

-mabo-

mabo on Tue Jan 25 14:11:28 2011 said:


My problems are:

1.) I have a really big pellet after the first step but after the first washing step, it seems smaller. Is this a sign of protein loss?

it could be due to loss of lipids

2.) I can´t resuspend my pellet in acetone for washing! The whole pellet is flying around. After mins of vortexing it falls apart, but in only ~10 pieces. Did I spin to fast/long?

try homogenizing with a dounce or a pellet pestle (if in an eppendorf tube)

3.) I can´t resuspend my pellet after drying! I tried different times from 5-30 min drying (room temperature or fume hood), so I exclude the possibility of overdrying the pellet. I tried to resuspend in the RIPA-buffer for quantification. Why does my pellet not resuspend?

what buffer are you using to resuspend? you should use sds-page loading buffer (without bromphenol blue if you want to quantify) if it is all going to be loaded on sds-page. it may require homogenization, as before

-mdfenko-

mdfenko on Mon Jan 31 23:55:41 2011 said:


mabo on Tue Jan 25 14:11:28 2011 said:


My problems are:

1.) I have a really big pellet after the first step but after the first washing step, it seems smaller. Is this a sign of protein loss?

it could be due to loss of lipids

2.) I cant resuspend my pellet in acetone for washing! The whole pellet is flying around. After mins of vortexing it falls apart, but in only ~10 pieces. Did I spin to fast/long?

try homogenizing with a dounce or a pellet pestle (if in an eppendorf tube)

3.) I cant resuspend my pellet after drying! I tried different times from 5-30 min drying (room temperature or fume hood), so I exclude the possibility of overdrying the pellet. I tried to resuspend in the RIPA-buffer for quantification. Why does my pellet not resuspend?

what buffer are you using to resuspend? you should use sds-page loading buffer (without bromphenol blue if you want to quantify) if it is all going to be loaded on sds-page. it may require homogenization, as before



Thank you very much for your reply!

1.) Im not lookin for lipids. So it doesnt matter?

2.) Yes, my sample is in an eppendorf tube. We do not own a "pellet pestle"...

3.) I tried to resuspend my pellet in RIPA-buffer, because of quantification and because it contains proteaseinhibitor. It also contains detergents, but in a lower concentration. Is protein quantification in SDS-page loading buffer (without bromphenol blue) possible?

Thank you very much!

-mabo-

mabo on Wed Feb 2 11:31:17 2011 said:


1.) Im not lookin for lipids. So it doesnt matter?

probably not. you can see if the protein content reduces to make sure.

2.) Yes, my sample is in an eppendorf tube. We do not own a "pellet pestle"...

a size 20 pestle should work or you could use a spatula or pasteur pipet or thin glass rod...

3.) I tried to resuspend my pellet in RIPA-buffer, because of quantification and because it contains proteaseinhibitor. It also contains detergents, but in a lower concentration. Is protein quantification in SDS-page loading buffer (without bromphenol blue) possible?

with proper blanking.

-mdfenko-