Generally other standard markers, like from Biorad or invitrogen, the first 2-3 bands are closer in 10% and again last 2 bands are close and this band distribution gives the location of b-actin band in about 45-50% lower region of membrane. However, in the case of SC-2361 marker-the b actin band location is relatively low (in 30-33% of lower portion of the Nitrocellulose membrane). I am worried if there is a technical mishap, or is it common to have different band heights for Actin in Nitro membrane in 10% gel running using different standard markers?
Please share your experience.
Hi,
I think it is just the way you measure the distance. Your santa cruz marker is broad range going from 200KDa to 6KDa. I am not sure what the range of your other markers is but I assume they are not the same range markers.
As you are using a very concentrated 10% gel, migration of the high KDa bands will be impeded. Therefore, measurement at this end of the curve will not give you the right size. If you are constructing the migration curve for the lower end of the size, you would need to use the result of your standards in the range of sizes that the gel is suitable for. That is, about 66KDA to 6KDA. This is why it is best to use markers that encompass the suitable range for your gel. If you know that with this standard the actin band runs at a certain spot, than you can just keep using this marker. But if you want accurate measurements, then use the right marker sizes. Hope it made sense.
Thanks, Pajkos, for your useful comment, well, even I did try another couple of braod range markers from Biorad and Santa both and results were still inconsistent. Out of frustration, one day I noticed the problem in one of my 1mL Pipette while I was making 10% gel...like 1mL was not dispensing full 1mL.....and I guess this lead to incorrect gel composition and thus migration of proteins was disturbed. Since I rectify this pipettes issue, there is a consistent B actin band corresponding with Santacruz broad range std marker. Anywayz, thanks once again for your time and efforts, best regards.
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