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

Irit Rappley via methods%40net.bio.net (by irappley from scripps.edu)
Sat Dec 17 16:46:40 EST 2011


Hi Sudheer,

Your protein stock is 1000 ug/mL = 1 ug/ul. That means you will need 32 ul protein stock to get your 32 ug sample. If the maximum volume you can load in your well is 16 ug, then you will not be able to prepare an appropriate 32 ug sample from the stock you have now. You can concentrate your protein stock if it's critical to get the 32 ug sample.

I'm not sure what is the calculation you are demonstrating, but the result does not make sense. If you were to take 0.128 ul from your 1 ug/ul (=1000 ug/mL) stock, then you would have only 0.128 ug protein. 

Using your 2X sample buffer, you could make the 0.25, 0.5, 1, 2, 4, and 8 ug samples. You will need more concentrated sample buffer (I usually use 5X or 6X) to load more protein. If you are using standard 1 mm protein gels, then I would guess that you can load 19-20 ul in each well if you are careful. That would allow you to make a 16 ug sample (16 ul stock + 4 ul 5X sample buffer, for example).

It's usually helpful to load the same (or at least similar) volume sample in each well. So for the 0.25, 0.5, 1, 2, and 4 ug samples, I would use 8 ul 2X sample buffer + Y ul sample + (8-Y) ul H2O, so that your final volume is 16 ul. 

Hope this helps.
Irit


On Dec 16, 2011, at 1:01 PM, sudheer sangeetham wrote:

> 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?????
> 
> Thank you in advance
> 
> -- 
> Sudheer Babu.S
> Ph.D student
> Institute of Biochemistry
> Biological Research Center
> Szeged,Hungary.
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