how to prepare protein sample, if I want to load exact amount on SDS-PAGE

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Sat Dec 17 13:34:18 EST 2011


In article <mailman.12.1324103754.3721.methods from net.bio.net>, 
sudheer.pbm07 from gmail.com says...
> 
> Hello Guys
> I have a trivial query, I have a protein stock 1000 µg per ml. Now I would
> like to load on each well 0.25, 0.5, 1, 2, 4, 8, 16 and 32 µgs.
> For 0.25  µg, 0.25  µl from stock + 0.25 µl 2x sample buffer , total 0.5 µl
> load on first lane. If I want to load higher amount like 16 µg and 32 µgs
> more volume to be loaded but I could load maximum16 µl in each well. In
> that case, should I use C1 V1 = C2 V2 formula, 1000 µg * ? = 16 µg *  8 µl
> V1 =  0.128 micro liter from the stock, 7.8 ul milli q water, to that add 8
> µl 2x sample buffer and load 16 ul to the well, Am I right doing things
> here?????

Your sample is 1 µg/µl. If your wells can hold only 16 µl and you still 
have to add the sample buffer, then the maximum sample size is 8 µl, 
which is equivalent to 8 µg of protein. The alternative would be to use 
gels with larger wells, so that you can load higher sample volumes.

What you should do in any case is to load the same volume into each 
well, otherwise the gel will run unevenly. So for example pipett 10, 8, 
6, 4, 2 µl sample into vials, make to 10 µl total volume with the buffer 
you have dissolved your protein in (i.e., 0, 2, 4, 6, 8 µl of buffer) 
and then add 10 µl of SDS-sample buffer to each sample. Mix and load 
your 16 µl per well. 


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