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Amount of protein loaded per well?

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#1 josbo

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Posted 26 May 2009 - 04:21 AM

Hi,
how much protein (in micrograms) is common to load to a 3o microliter well. I've seen suggestions from 20 to 200 microliter, but what's the standardamount?

#2 yean_ny_nie

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Posted 26 May 2009 - 04:28 AM

Hi,

Before determining the amount of protein loaded, did u specified the protein concentration by running a protein test such as BCA Assay? The amount of protein load into a well can be determined from the research you are doing. For my case, I'm running a SDS-PAGE with 20 to 60ng of protein with total volume of 20ul (protein sample + dissociation buffer + dH2O).

#3 little mouse

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Posted 26 May 2009 - 04:30 AM

For a cell extract, I load around 20-30 µg. for a purified protein, I load much less (50 ng, depending on the antibody too)

#4 marfly

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Posted 26 May 2009 - 05:00 AM

I've found that for purified protein 5 µg gives a nice band. For cell cultures (e.g. uninduced and induced culture) I calculate the appropriate amount to load based on the OD at 600 nm: If I pellet a 1 ml sample at OD=0.8 I resuspend it in 70 µl buffer/medium/water, mix 15 µl of it with 5 µl 4x loading buffer and load all. For OD=3.5 (overnight induction) I resuspend the 1 ml pellet in 500 µl and do the same. That gives nice comparable samples on my gel (although I don't know if this is the proper way of doing it... ).

#5 josbo

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Posted 27 May 2009 - 12:18 AM

Hi,

Before determining the amount of protein loaded, did u specified the protein concentration by running a protein test such as BCA Assay? The amount of protein load into a well can be determined from the research you are doing. For my case, I'm running a SDS-PAGE with 20 to 60ng of protein with total volume of 20ul (protein sample + dissociation buffer + dH2O).

Thanks for answering! I did a BCA assay and also I measured the protein concentration on the Nanodrop. Do you mean nanograms or micrograms? What dissociationbuffer do you use? Additional things like DTT, urea or beta-mercapto? How much dissociationbuffer, water and sample in your 20 microliter mix?