I am a newbie in 2D gel electrophoresis, my aim is to see the protein expression profile of a bacterium when grown in cold temperature and 37 degrees?
For 2D gel electrophoresis, the protein extraction is very important. The buffer that you will use should be done with 2D electrophoresis quality products such as urea or thiourea. It is very important that you remove the salt in your protein extract. I was using desalting kit from Biorad to optimize the first dimension separation.
Googd luck
Johan
If I understand your question correctly, you want to compare the protein profiles from two groups of bacterium, one growths in cold temperature and the other growth in 37 degree.
Base on my experience, sample preparation is important for conducting 2D gel electrophoresis. Therefore, you could tried to look up some previous reports. If other already counted the experiment using the bacterium that you about to use, you could start by trying to follow their protocols.
Good luck,
Hao-Wei
For 2D gel electrophoresis, the protein extraction is very important. The buffer that you will use should be done with 2D electrophoresis quality products such as urea or thiourea. It is very important that you remove the salt in your protein extract. I was using desalting kit from Biorad to optimize the first dimension separation.
Googd luck
Johan
In the attached pdf (2-D Electrophoresis Principles and Methods from Amersham Biosciences) you will find a wealth of useful inormation that will help you to set up your 2D electrophoresis experiments.
Best regards
We use the following protocol for 2D Gel Electrophoresis... Hope it works for you.. Good Luck
If you dont have suitable equipment for IEF, it could be helpful to use native electrophoresis in the first dimension and in the second SDS Page. Have look at attachment. Good luck
thank you every one for their valuable suggestions and solutions.. i will work upon these and if i come across queries, i will post further... thanking you once again....
Based on my experiences for 2D electrophoresis, it is better to use protein samples after cold acetone precipate which help in remiving all salts and debris from the samples as 2D require high quality protein samples and it is better to run 1D gel in order to confirm presences of sufficient amount of protein in samples and of course you have to measure it before start of your work
- Badges
- Science method