sample preparation for SDS PAGE - (Dec/12/2007 )
hye..i want to ask about maximun volume of protein to run in SDS PAGE gel.is it ok if the volume of the sample was different from others sample run togather in the gel..but all the samples have same concentration(20ug of protein)
is any one here have used laemmli sample buffer from biorad???how the sample preparation?is it the protein sample and the sample buffer was 1:1? pls hepl me
tq
hi
I use bio-rad sample buffer. i found its contants in their site.
u should use it 1:1 with the protein, meaning 2XSDS-PAGE, but first u have to add
beta-mercapto (1:20 ratio) or DTT
I agree with Amtash on the BioRad Sample Buffer, 1:1 with sample, although it's not a critical thing, adding more also works. Regarding the volume matter, the important thing is that you load the same amount of protein for all your samples if you want to do direct comparisons, it doesn't matter if the resulting volumes are different. The maximum volume will depend on the capacity of the wells you are using...
we have found that there are enough times (not always) where volume does matter that we adjust all of our samples to the same volume.
how can we adjust the volume?is it we have to used our elution buffer that we used for the protein sample.i have read from protocol from internet that suggest us to adjust the volume using water or PBS..is it ok to used water or pbs or we should used our same buffer for our sample..
In our lab, we use a 6x loading buffer that we make. So we determine protein content of our extracts with the Bradford method, take the amount of extract (35ug), add the 6x buffer to 1 x final and fill the mix with water to 50 ul. So I have the same volume and concentration for all the tracks.
Different volumes with the same loading buffer and the same concentration should be fine, though.
we use water to avoid adding more salt to the sample but, to keep all things equal it would be better to use the buffer that your samples are in (if they are all in the same buffer, if not then water is your best bet).
we use water to avoid adding more salt to the sample but, to keep all things equal it would be better to use the buffer that your samples are in (if they are all in the same buffer, if not then water is your best bet).
Do you adjust the volumes to get bands of same size (especially width)? This problem has been bothering me for a long time. I didn't even think about it.
yes, we found that it helps to reduce encroachment.