4 replies to this topic

[cardosopedro]

Hi.

I want to concentrate my cell lysates for western blot by precipitation of the proteins with trichloroacetic acid + acetone. I have some questions.

1. This treatment will not modify my proteins for the posterior immunoblot detection?

2. After the precipitation, what is the best method to store the lysates? Can I freeze the pellet? Or should I ressuspend it in some buffer or H2O and store it like that?

3. Can I quantify the protein concentration by the Bradford assay (as I usually do)?


Thanks in advance.

[Prep!]

1) ya you can do a WB.. it sure will denature your protein but preferably use a polyclonal Ab and u shud not have a detection problem.
2) i am not sure about this but it is preferable to solubilise it and then store.
3) Yes you can. IT wont do anything to the basoc amino acids which is needed for the quantitaion by bradford

[mdfenko]

you may have trouble solubilizing the pellet. most solubilize with loading buffer after washing out residual tca.

[cardosopedro]

Thanks for both answers.

So I solubilize the pellet in loading buffer and then I can freeze the samples like this?

[mdfenko]

cardosopedro, on Nov 20 2009, 05:41 PM, said:

Thanks for both answers.

So I solubilize the pellet in loading buffer and then I can freeze the samples like this?

yes, but it may be better to heat denature then freeze.