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SDS-PAGE Gel Electrophoresis Forum SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


gel loading question

gel loading question - SDS-PAGE Gel Electrophoresis Forum

gel loading question - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


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Old 03-24-2010, 01:38 AM
tgd tgd is offline
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Default Re: gel loading question

Hello,

Well, if your protein concentration is 0.1ug/ul and you require 15ug to be loaded per track you will have to concentrate the sample. Say a 10-fold concentration (to about 1 ug/ul) and load 15ul.

You have not said how much you have volume-wise, or whether the sample is in sample buffer containing SDS.

If the protein is in the native state, you can concentrate using a Centricon. These have a semipermeable membrane with various molecular weight cut-offs (commonly 10 000 kDa), and concentration is achieved by centrifugation. You will easily concentrate 1 ml to, say, 100 ul by this method in about 20 min.

A second possibility it to precipitate with trichloroacetic acid (TCA). One protocol is to add an equal volume of 20% TCA, leave on ice for 20 min and then centrifuge for 10 min at (say) 10 000 g. There are two problems. Firstly, the method is NOT quantitative and, secondly, it can be very difficult to get the TCA pellet to dissolve in SDS sample buffer.

I prefer the Centricon method. However, as the previous post said, it is better to design your extraction/experiment in such a way that the protein is obtained in a concentrated state (1mg/ml or higher).

Good luck,

tgd
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