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To check a protein expression I pelleted a small amount of E. coli before and after induction and lysed them by redissolving them in SDS-PAGE loading buffer and heating them to 95 °C for 1 minute.

This lead to a solution with some very sticky and viscous parts in it, that make pipetting the sample into the gel wells extremely annoying. As far as I heard, this is probably genomic DNA, and my usual way to deal with this is to centrifuge the samples and only pipet a small part out from the top. This does seem to help sometimes, but not always.

How can I avoid the formation of that sticky and viscious stuff or how can I avoid pipetting that stuff into my wells?

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Just to say I have the same problem! So I look forward to the answer. –  Ben Jun 28 '12 at 17:15

2 Answers 2

up vote 6 down vote accepted

Yes, it is the genomic DNA that is causing you trouble. Although a brief 1000g spin should bring it all down, the pellet is never tight and you almost always are going to pull up some gunk with the clear supernatant. A better solution is to include a quick sonication step (5-10s) before the 1000g spin. That way, the DNA is sheared and it should pellet better. It has worked for me most times. Also, remember to use a reasonable volume of SDS buffer when you lyse the cells. Too little buffer and its always going to be hard. For comparison, I use about 200ul per 1ml culture pellet and this volume works well for both pre- and post-induced cells.

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In addition to gkadam's suggestion, for my protein gels, I heat my samples at 95C for 10 minutes. I would also look into different lysing conditions. Rather than using SDS loading buffer, look into more vigorous lysing steps either with a kit like bug-buster or using a homogenizer. When pelleting your lysate, I've traditionally done a 10,000g spin for 10 minutes.

Maybe I just work with really soluble proteins...

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Definitely your suggestions will make things much better. I think Mad Scientist was looking for a quick and dirty way to do it when doing small scale checks for protein expression from a new construct. –  gkadam Jul 1 '12 at 0:46

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