GST fusion protein preparation troubleshooting..

[xBobbyCx]

Hi all,

 

I require soem help in my experiment.. Did a few round already, cos the first round, my protein was not pure ie cross contamination between V8-GST adn GST protein. 2nd round was low yield.. 3rd round (the most recent) the protein sort of degraded a little bit.

 

But I made sure not to cross contaminate by using loads of pipettes.. And I made sure to put my protein samples on ice all the time. And centrifuging at 4 degrees when i need to wash off excess protein bound to beads. What could cause all possible errors?

 

Could soemone pls help with troubleshooting, ie suggest possible steps in which i could hv gone wrong??

 

these are the steps i really paid attention to:

1. prevent cross-contamination by using different pipettes

2. put samples on ice all the time

3. use fresh elution buffer and lysis buffer

4. sonicate so that cell culture become clear

 

thank you!

[Prewitt]

just did that twice...but

1.how did you notice you protein was degraded? --> see if buffer ph and omsomlarity are optimal

2.to avoid contamination: wash more.

3.gst is a dirty thing. it simply is.