Western Blot of Bacteria - Need some advice on performing a Western Blot from Bacteria (May/06/2008 )

Hi! I'm new here, and I have a few questions regarding performing a Western blot from bacteria:

Once antibodies are made for me, I will be performing a Western blot on Legionella pneumophila. I've never done any protein work in my life before.

While I am waiting for the antibodies to arrive in a month or so, I would like to prepare the bacteria by growing them in broth to mid-late log phase (sound correct so far?). I would then like to pellet an aliquot of the cell culture (I'm assuming 1-2 ml) and then freeze it for lysis at a later date. My question for right now is regarding the preparation and freezing of the bacterial cell pellet (at -70C), with the growth medium removed of course. Should I resuspend the pellet in some type of a buffer? (1x PBS perhaps) Or, can I just freeze the pellet alone without resuspending at -70C?

If anyone has any articles/protocols that would be of great help to me.

Thank you for your time to anyone who contributes.

I used to resuspend the pellet in laemmli buffer directly. It's the best way to solubilize most of the proteins.
However, in these conditions you can't do a protein titration.

The second result has an answer for you. I think it should be same for all bacteria, can't be sure though.

http://search.vadlo.com/b/q?sn=158621799&a...stern&rel=0

HTH/

While I am waiting for the antibodies to arrive in a month or so, I would like to prepare the bacteria by growing them in broth to mid-late log phase (sound correct so far?). I would then like to pellet an aliquot of the cell culture (I'm assuming 1-2 ml) and then freeze it for lysis at a later date. My question for right now is regarding the preparation and freezing of the bacterial cell pellet (at -70C), with the growth medium removed of course. Should I resuspend the pellet in some type of a buffer? (1x PBS perhaps) Or, can I just freeze the pellet alone without resuspending at -70C?

If anyone has any articles/protocols that would be of great help to me.

Thank you for your time to anyone who contributes.
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In my experience you can store at -20°C or -70°C the pellet from 1ml of colture if you want to store them for a month before resuspending each pellet in lysis buffer, with the aim to separate the soluble fraction from the insoluble one. In this case, you can ceck the protein concentration in the lysate vith Bradford or BCA protocol. But if you do not need to separate the soluble proteins from the whole lysate you should prefer to resuspend each pellet in sample buffer, proportionately to the colture OD. This way is simplier and faster, you have no need to disrupt the cell with sonication or other, just centrifuge and resuspend colture in sample buffer, boil samples e load them on SDS-page for western blot!