purification of proteins from sample - (Apr/27/2007 )
I have alot of sample (~150 ml) from which I want to analyze the protein content by gel electerophoresis. Is there a smart strategy for upconcentrating the proteins without losing any of them. I was thinking about acetone precipitation, but I have never done that with sample volumes over 1 ml.
-kottila-
QUOTE (kottila @ Apr 27 2007, 04:02 AM)
I have alot of sample (~150 ml) from which I want to analyze the protein content by gel electerophoresis. Is there a smart strategy for upconcentrating the proteins without losing any of them. I was thinking about acetone precipitation, but I have never done that with sample volumes over 1 ml.
1. Depends on Mw - concentration on microcon by ultrafiltration or ultrafiltration on Amicon cell
2. TCA precipitation work well and standart technique for concentration proteins for PAAG
3. Increase sensitivity of gel staining with silver staining technique
-circlepoint-
QUOTE (circlepoint @ Apr 27 2007, 03:01 PM)
QUOTE (kottila @ Apr 27 2007, 04:02 AM)
I have alot of sample (~150 ml) from which I want to analyze the protein content by gel electerophoresis. Is there a smart strategy for upconcentrating the proteins without losing any of them. I was thinking about acetone precipitation, but I have never done that with sample volumes over 1 ml.
1. Depends on Mw - concentration on microcon by ultrafiltration or ultrafiltration on Amicon cell
2. TCA precipitation work well and standart technique for concentration proteins for PAAG
3. Increase sensitivity of gel staining with silver staining technique
The Mw is totally unknown. I just want an overview of which proteins are in t he sample. Isn't TCA precipitation a bit difficult with large volumes (i.e. having a tiny protein precipitate in a large container?)
-kottila-
QUOTE (kottila @ Apr 27 2007, 05:14 AM)
QUOTE (circlepoint @ Apr 27 2007, 03:01 PM)
QUOTE (kottila @ Apr 27 2007, 04:02 AM)
I have alot of sample (~150 ml) from which I want to analyze the protein content by gel electerophoresis. Is there a smart strategy for upconcentrating the proteins without losing any of them. I was thinking about acetone precipitation, but I have never done that with sample volumes over 1 ml.
1. Depends on Mw - concentration on microcon by ultrafiltration or ultrafiltration on Amicon cell
2. TCA precipitation work well and standart technique for concentration proteins for PAAG
3. Increase sensitivity of gel staining with silver staining technique
The Mw is totally unknown. I just want an overview of which proteins are in t he sample. Isn't TCA precipitation a bit difficult with large volumes (i.e. having a tiny protein precipitate in a large container?)
Why large volume but not simply 1 ml aliquote??
-circlepoint-
QUOTE (circlepoint @ Apr 27 2007, 03:42 PM)
QUOTE (kottila @ Apr 27 2007, 05:14 AM)
QUOTE (circlepoint @ Apr 27 2007, 03:01 PM)
QUOTE (kottila @ Apr 27 2007, 04:02 AM)
I have alot of sample (~150 ml) from which I want to analyze the protein content by gel electerophoresis. Is there a smart strategy for upconcentrating the proteins without losing any of them. I was thinking about acetone precipitation, but I have never done that with sample volumes over 1 ml.
1. Depends on Mw - concentration on microcon by ultrafiltration or ultrafiltration on Amicon cell
2. TCA precipitation work well and standart technique for concentration proteins for PAAG
3. Increase sensitivity of gel staining with silver staining technique
The Mw is totally unknown. I just want an overview of which proteins are in t he sample. Isn't TCA precipitation a bit difficult with large volumes (i.e. having a tiny protein precipitate in a large container?)
Why large volume but not simply 1 ml aliquote??
Because of the low concentration of proteins. I want to up concentrate the samples before running on gel
-kottila-
QUOTE (kottila @ Apr 27 2007, 05:45 AM)
QUOTE (circlepoint @ Apr 27 2007, 03:42 PM)
QUOTE (kottila @ Apr 27 2007, 05:14 AM)
QUOTE (circlepoint @ Apr 27 2007, 03:01 PM)
QUOTE (kottila @ Apr 27 2007, 04:02 AM)
I have alot of sample (~150 ml) from which I want to analyze the protein content by gel electerophoresis. Is there a smart strategy for upconcentrating the proteins without losing any of them. I was thinking about acetone precipitation, but I have never done that with sample volumes over 1 ml.
1. Depends on Mw - concentration on microcon by ultrafiltration or ultrafiltration on Amicon cell
2. TCA precipitation work well and standart technique for concentration proteins for PAAG
3. Increase sensitivity of gel staining with silver staining technique
The Mw is totally unknown. I just want an overview of which proteins are in t he sample. Isn't TCA precipitation a bit difficult with large volumes (i.e. having a tiny protein precipitate in a large container?)
Why large volume but not simply 1 ml aliquote??
Because of the low concentration of proteins. I want to up concentrate the samples before running on gel
Concerning pricipitation I think that it is not convenient for you because of different patterns of protein precipitation will occur and if you work in low concentration level it will be a problem and so you could not estimate correctly all range of your proteins in precipitate!
Concerning Amicon concentration Mw cutoff 3kDa is not enough for your range of interest?
what is about silver staining? It is good enough for your purpose
-circlepoint-
Three Phase Partitioning
1. add t-butanol to your protein solution to get 20% of concentration
2. add solid ammonium sulphate to get 100% of saturation and stirr for an hour
3. centrifuge at 5000-10000*g for 10-30 minutes
Protein would form thin layer between two solutions. We tried it in my lab and it works fine for polypeptides from 5kDa to 150kDa. According to papers where I found it, this method is safe for proteins, it can even re-naturate some proteins. They also say that protein layer do not contains much salt, so it can be used for ion exchange chromatography without destalting.
-K.B.-
QUOTE (K.B. @ Apr 27 2007, 08:06 AM)
Three Phase Partitioning
1. add t-butanol to your protein solution to get 20% of concentration
2. add solid ammonium sulphate to get 100% of saturation and stirr for an hour
3. centrifuge at 5000-10000*g for 10-30 minutes
Protein would form thin layer between two solutions. We tried it in my lab and it works fine for polypeptides from 5kDa to 150kDa. According to papers where I found it, this method is safe for proteins, it can even re-naturate some proteins. They also say that protein layer do not contains much salt, so it can be used for ion exchange chromatography without destalting.
1. add t-butanol to your protein solution to get 20% of concentration
2. add solid ammonium sulphate to get 100% of saturation and stirr for an hour
3. centrifuge at 5000-10000*g for 10-30 minutes
Protein would form thin layer between two solutions. We tried it in my lab and it works fine for polypeptides from 5kDa to 150kDa. According to papers where I found it, this method is safe for proteins, it can even re-naturate some proteins. They also say that protein layer do not contains much salt, so it can be used for ion exchange chromatography without destalting.
Thank you K.B.!
Very interesting. Could you give citation on original source
-circlepoint-
QUOTE (kottila @ Apr 27 2007, 01:02 PM)
I have alot of sample (~150 ml) from which I want to analyze the protein content by gel electerophoresis. Is there a smart strategy for upconcentrating the proteins without losing any of them. I was thinking about acetone precipitation, but I have never done that with sample volumes over 1 ml.
have a trial with non-denaturizing ammonium sulphate precipitation subsequently followed by dialysis with correct cut-off
-The Bearer-
QUOTE (kottila @ Apr 27 2007, 01:02 PM)
I have alot of sample (~150 ml) from which I want to analyze the protein content by gel electerophoresis. Is there a smart strategy for upconcentrating the proteins without losing any of them. I was thinking about acetone precipitation, but I have never done that with sample volumes over 1 ml.
I'd recommend to use Centricon or Centriplus (whichever is suitable, I can't remember) concentrators. Check out Millipore's website to find the right one or Vivaspin is another brand by Sartorius (I'm not sure, but I think it is Sartorius).
They've got low cut-off stuff (~ 3000 Da or probably even less).
-Krisztina-