3 replies to this topic

[mabo]

Hi all!
I´m a student from germany!


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

I use RIPA-buffer for cell lysis:

Quote

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 µg/ml Pepstatin
50 mM sodium fluoride (NaF)
1 mM sodium orthovanadate (NaV)
1 mM p-nitrophenyl phosphate (PNP)
1 mM Na-ß-glycerophosphate

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

Then I follow this protocoll:

Quote

1.) Centrifuge the sample(s) at 15.000xg 10 minutes at 4°C.
2.) Discard the supernatant properly.
3.) To the pellet add 800 µl 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 can´t 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 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?

I will be very thankful for any help!

Greetings!

[mdfenko]

mabo, on 25 January 2011 - 06:11 AM, 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

Quote

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)

Quote

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

Edited by mdfenko, 31 January 2011 - 03:57 PM.

[mabo]

mdfenko, on 31 January 2011 - 03:55 PM, said:

mabo, on 25 January 2011 - 06:11 AM, 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

Quote

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)

Quote

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

Thank you very much for your reply!

1.) I´m not lookin for lipids. So it doesn´t 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!

[mdfenko]

mabo, on 02 February 2011 - 03:31 AM, said:

1.) I´m not lookin for lipids. So it doesn´t matter?

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

Quote

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...

Quote

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.