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Equal Loading Confusion

SDS-PAGE

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6 replies to this topic

#1 CarlosJ

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Posted 09 June 2013 - 12:09 PM

Hi,

Does 'equal loading' on each SDS-PAGE wells means equal sample volume (e.g. 10 ul / lane) or equal amount of protein (e.g. 20 ug / lane)?

Thx.

#2 bob1

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Posted 09 June 2013 - 12:42 PM

Usually equal loading of protein content.

#3 mdfenko

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Posted 10 June 2013 - 03:59 AM

but you should also equalize volumes to avoid lane artifacts
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#4 CarlosJ

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Posted 10 June 2013 - 08:07 PM

Usually equal loading of protein content.

Thanks bob1.

but you should also equalize volumes to avoid lane artifacts


mdfenko, do you mean i've to equalize both content (in ug) and volume (in ul)? Then in that case, I would have to dilute my samples to equalize the sample concentrations because my samples have different concentration. For example, from Bradford Assay, Sample A conc. = 2000 ug/ml ; Sample B conc. = 2500 ug/ml ; Sample C conc. = 3000 ug/ml
So do I have to equalize all my samples to the lowest sample concentration, in this case Sample A concentration of 2000 ug/ml? If yes, with what do I dilute my samples?
I've seen protocols which mention that we need to mix our samples with SDS-PAGE sample buffer to at least 1:2 proportion. If i've to equalize all my samples concentration, do I do the equalization before or after mixing the samples with sample buffer at 1:2 proportion?

Thx.

#5 bob1

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Posted 11 June 2013 - 01:19 AM

You could do it the way you suggested, or you could take out a certain amount (say 20 ug from each) and then dilute those to the same volume. I would dilute in the lysis buffer, with appropriate amount of sample buffer added.

#6 mdfenko

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Posted 11 June 2013 - 04:19 AM

as bob1 says, dilute with the buffer in which you have the samples (assuming they're all the same). then add the appropriate amount of sds sample buffer for the volume of the diluted samples (this way, all of the samples should be under identical conditions in the gel).

of course, you won't be diluting the whole preparation, just an aliquot with the appropriate amount of protein for the gel.

Edited by mdfenko, 11 June 2013 - 04:21 AM.

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#7 CarlosJ

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Posted 16 June 2013 - 07:40 AM

Thank you so much, bob1 and mdfenko. Your explanations cleared up my confusion now :-)





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