Western Blot of cells in Laemmil - (Oct/09/2007 )

Hi there,

I have a question. I have to do a Western Blot of cellculture cells which are lysed with Laemmli buffer (200 µl per well in a 6 well plate). Usually I immediately use this extract (20 µl) for Western Blot. The problem is that today all the Western Blot equipment is in use by someone else but I need the samples to be taken today. Can I freeze it and do the Western tomorrow? Any tips?

Stardust

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Sure you can freeze the sample inLaemli buffer and run the SDS-Gel later. That is no problem. you also could store it at 4°C as Laemmli denatures all proteins and there should be no protease activity left in the samples. But with frozen samples I have made good experience.

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Thanks for your help! I have one more question: do I have to boil them before freezing or after thawing or both?

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better is to shock-freeze the lysats w/o Laemmli-buffer; freezing with Laemmli-buffer may result in lesser sharp bands;

a short re-boiling is useful but not necessary

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Boil the samples as soon as you have the lysate with laemmeli buffer. After boiling, your sample is stable at 4 degree. If you are going to run the gel any time soon (within about a week) I would recommend 4 degree. As mentioned, some people say freezing the sample will make the bands worse but I've never had an issue. I always re-boil for one minute after thawing. This helps make sure all the SDS is in solution.

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Thanks to everyone!

Stardust

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