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Easy method to lyse E. coli pellet (follow-up of GST fusion protein purification


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#1 sonjalin

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Posted 06 June 2012 - 09:48 AM

Hi,

this is very basic question, but haven't worked with bacterial cells that much...

I'm performing GST fusion protein purification and I have a good protocol for that. I'm taking an 1-2 ml culture sample before and after IPTG inducing which I'll run on SDS PAGE to be able to see that the inducing works. What's the easiest way to lyse these cell pellets? Sonicator is not in the same building so wouldn't like to use that.

#2 Papaver

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Posted 07 June 2012 - 02:31 AM

If you only want to check for protein expression, pellet the culture sample and resuspend the pellet in SDS-loading buffer. The amount depends on the size of the pellet. I usually resuspend the "before-sample" in 100 - 150 µl loading buffer and the "after-sample" in 300 - 450 µl. Then heat the samples for 5-10 min at 95-99 °C and load 15 µl of "before" and 7,5 µl of "after" onto SDS-PA gel.

#3 sonjalin

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Posted 07 June 2012 - 07:25 AM

I've tried that and today it was quite fine - although there's still blue "background" on a gel and bands are not that easy to detect. First time I didn't centrifuge down after boiling so the background was so high that I couldn't detect anything...

Today I resuspended 2 ml "before" sample (=OD590 was about 0,5) in 50 ul of loading buffer and 2 ml "after" sample in 90 ul if I remember right. Then I centrifuged 15 min 13 000 g and loaded on gel about 20 ul of the supernatant. Bands weren't that strong - but the blue background caused difficulties. Should I load less and/or resuspend in larger volume? The background is perhaps because of DNA?

#4 almost a doctor

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Posted 07 June 2012 - 08:09 AM

What do you mean by blue background? You should see loads of bands of different MW, and if your expression is working on of those bands (around the MW of your expected protein) will be much stronger.

I used to take 1ml of culture before, and 1ml after induction, spin down, remove media, resuspend in 100-200ul of SDS-Buffer, boil and load on gel. Sometimes this preps are very gloopy and hard to load, a way round this is to put your samples (after resuspending in SDS-buffer) in the -80C freezer for a bit (~30min should do the trick, you can also snap freeze them if you have access to dry ice or liquid N). After this just boil, straight from the freezing, and proceed the same way a before.

#5 Papaver

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Posted 07 June 2012 - 09:34 AM


Today I resuspended 2 ml "before" sample (=OD590 was about 0,5) in 50 ul of loading buffer and 2 ml "after" sample in 90 ul if I remember right. Then I centrifuged 15 min 13 000 g and loaded on gel about 20 ul of the supernatant. Bands weren't that strong - but the blue background caused difficulties. Should I load less and/or resuspend in larger volume? The background is perhaps because of DNA?


I would not do it like you did...just pellet the cells and resuspend them in SDS loading buffer. If it it still "gloopy" you can also add more loading buffer. Try it the way I wrote in the first comment...so far it always worked Posted Image

I guess your "backround" is because you overload the....20 µl is too much. Do you destain your gel with water properly after Coomassie-staining?




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