Hi
i am using a molecular weight marker from Bangalore Genie (India)(Old catalog no. PMWM, new catalog no. 105979). In the image below A is the pattern which the company has given in the catalog, whereas B is the pattern which I got on same- 12% SDS-PAGE. As you can see clearly, there is a difference in the spacing between bands of the 2 patterns? Can anyone comment on why is it so? Is such case can I just count number of the bands and then compare the pattern order wise?

Hi Ram,
The spacing depends on the gel percentage and for how long the gel was run....more the run time, more will be the resolution. Similarly on a lower percentage gel, the upper area will be expanded........to compare with ur gel, u will hv to count the no. of bands....

Your spacing looks appropriate for a 12% SDS-PAGE gel, based on the molecular weights of the markers.

But both A and B are 12% PAG, then why is there such difference !?

Maybe the lot you received is different than what is printed in the catalog.

Is it just my imagination, or does band 3 on the right have a doublet?

hi ram... don mind but its bangalore genie!!!
this is not a surprice to me... and ya jsut possible that they boiled for less time you for more.. or something like that.. i mean sample treatent might hav been different!!!
or as lab rat says it might be a defective lot also!!!
i wuld not just count the bands and go ahead as the saing does not look okay to me given the molecular weights...

Yah.. there is a doublet in band # 3. If this is counted as 2 bands, then there are 6 bands in A and 7 in B!
Pradeep...does the inconstant pre-loading boiling really results in such a variable results?

well, i don't think so........such variability, due to boiling, i doubt???? y dont u try NEB ladder

well i was telling that cause i have observed (not with marker) with some proteins, that under reduced conditions, even the boiling time lead to difference in the migration pattern!!!
But my stress was on the sample preparation.. boiking was jsut an example

because I dont have it right now!

Just plot each protein weigh vs mobility you will probably obtain a curve intead of a straight line, that's ok.

It seems that you got higher resolution of the lower weight proteins this may be due to:
-The inclusion of urea or glicerine in the gel.
-Different buffer in the + and - pole
-Reusing buffers.
This is usually an effect of different molarity between the gel buffer and +pole buffer.
By the time the gel is inmersed in the tank, if your gel has lower molarity than the tank buffer it will start to slowly equilibrate by more molecules (Tris probably) entering the gel. This will increase the resistance and thus current on that end of the gel, thus your proteins will run faster on the border of the gel.

If you want to be sure the marker is alright, which it probably is, run BSA or other know weight protein on a parallel lane. Keep the same salt, SDS-detergents, protein qt content on each lane.

Assuming the ladder actually contains the proteins it's supposed to (and thus they are of known molecular weight), the pattern comparison between your gel and the company's gel is irrelevant -- if that is how a 20 kDa protein runs on your gel, then a 20 kDa protein from your sample will run the same way on that gel, and the comparison of your sample to the ladder is valid for estimating the relative molecular weight.

Hi!! Ram,
I think from my experience your pattern appears more appropriate for a 12% gel. The Catalogue pattern seems to be of 9%or may be even 7.5%...
I think you lodge a complaint...

It may be also that you and the company use different acrylamide stocks, lets say you use 19:1 and the company 37.5:1 that would also explain it.