8 replies to this topic

[Margoute]

Hi all,

I ran a SDS-PAGE, using Kaleidoscope Prestained Standards from BioRad as a molecular weight marker.
I got a nice band for my protein - only visible band. But the problem was - it appeared about 80kDa lower than I expected - the protein is supposed to be 160 kDa, but it showed up as 80kDa, according to the MW marker.

I called the company, they suggested a few possible reasons for that - like degradation of the protein or MW of 160kDa for  the protein being in dimers, which as I think, is not the issue.

They sent the positive control (HepG2 cell lysate), and meanwhile i ran another gel, from another kind of tissue, probing for a different, unrelated protein - and it showed up as being 40kDa, instead of 120kDa.

I re-ran the first gel, with the positive control, and the positive control showed the most intensive band around 80kDa, together with the most intensive bands from my samples, so I think there should be something wrong with the MW marker?

What could be a reason for this?

Thank you

Edited by Margoute, 09 March 2009 - 08:10 AM.

[Dr Teeth]

Simplest test--try new markers.  
How about B-actin or other controls?  Is anything running at the expected MW or are all of them 80 kD off?  
Are you transferring all of the markers?  Oftentimes, the highest MW markers do not fully transfer from gel to membrane, but should still be taken into account when calculating MW of proteins on the membrane.
Also, if you can show a scan of your blot, that would be helpful.

I have not had any trouble using those same markers in conjunction with prepoured gels from Invitrogen.

Edited by Dr Teeth, 09 March 2009 - 10:47 AM.

[butterfly]

Margoute, on Mar 9 2009, 08:09 AM, said:

Hi all,

I ran a SDS-PAGE, using Kaleidoscope Prestained Standards from BioRad as a molecular weight marker.
I got a nice band for my protein - only visible band. But the problem was - it appeared about 80kDa lower than I expected - the protein is supposed to be 160 kDa, but it showed up as 80kDa, according to the MW marker.

I called the company, they suggested a few possible reasons for that - like degradation of the protein or MW of 160kDa for  the protein being in dimers, which as I think, is not the issue.

They sent the positive control (HepG2 cell lysate), and meanwhile i ran another gel, from another kind of tissue, probing for a different, unrelated protein - and it showed up as being 40kDa, instead of 120kDa.

I re-ran the first gel, with the positive control, and the positive control showed the most intensive band around 80kDa, together with the most intensive bands from my samples, so I think there should be something wrong with the MW marker?

What could be a reason for this?

Thank you

I had similar problem before. I even tried to run markers from different company, bio-rad and NEB, side by side. They all runed same, but my protein size is incorrect. Later, I figured out the problem is from the running buffer. I make a new batch of running buffer, then, my protein run to the right size! So, you may want to test if your running buffer is too old

Good luck!

[mikew]

Have other people blotted for this protein?
Is it a commercially available antibody?
If this antibody is commercially available the most reaonable explanation is simply that your western blot didn't work.
The markers will not be that "off". I have used BioRad markers of all types for many years and have found them to be the best on the market.
Most likely your antibody is detecting a non-specific band.

[HomeBrew]

Colored molecular weight markers are not useful for accurate size determination, only for monitoring the run or western transfer efficiency.  Due the dyes covalently bonded to the proteins, their apparent molecular weight changes, sometimes dramatically, due to differing run conditions, like buffer and gel composition, pH, etc.

This fact is well known; it's why colored markers are always shown with "apparent" molecular weights for the bands.  For example, see Q3 here, or this .pdf from Invitrogen, or just Google "prestained markers apparent molecular weight".

I'm surprised the BioRad rep wouldn't mention this...

For accurate size determination, use an unstained ladder, and stain the gel.

[mikew]

Homebrew,

Yes that is correct. Colored markers are often off by 10-30 kDa. However,
No Marker that runs at 160 kDa should be showing up at 80 kDa.
If a protein is 180 kDa and all that is seen on the Western is a band at 40 kDa
this is simply not a problem of the marker.
  Has the person done any positive controls? For example, how does p53 stack up to this ladder?
If proper controls were done and the proteins are appearing to be running at aberrant weights,
then the gel is improperly made.
I have used practically every protein ladder known to man and I have never seen a protein run more than 40 kDa off its expected weight with a colored marker.
Even then, that's a stretch.

[HomeBrew]

Perhaps.  But we won't know how far off anything is until the protein of interest is run against an accurate standard.  Perhaps the protein has a signal sequence cleaved from it, or it's being subjected to protease cleavage, or the start codon is called wrong -- there ar any number of reasons the recovered sample could actually be smaller than the 160 kDa that it's "supposed to be".  Couple this with an inaccurate standard, and it could easily explain the results seen...

On the table in the link I provided to NEB, there's a ~50 kDa swing in the largest band, depending on the conditions, and on the Invitrogen table, there's a couple of ~60 kDa swings in the larger bands of the marker.

[Margoute]

Thank you all for promt and useful answers,

Dr.Teeth:
I will be reprobing for B-actin today, maybe this alone will clear the question, and yes, all the markers transferred, they all are of different colors, so I would notice if any was missing.

Butterfly:
-the running buffer was fresh, and I will re-make it, just in case something wrong was in making it

Mikew:
I searched the literature, and other people did not seem to have problem with it – their western blot figures show this protein to be at 160kDa, and the antibody is commercially available… but its hard to imagine that the antibody picked the wrong band – the blot has only one nice intensive band – in both cases with 2 different antibodies to probe for 2 different proteins for 2 different projects, with 2 different MWs but with a similar “error” of 80kDa between reported and determined MW.

Homebrew:
I checked the links, and I think this can be an explanation for the problem, I searched the web, and it seems to be the most possible reason, also – it could be the gel itself – as mikew also pointed out.
I also thought of possible protein modifications, as you said, I don’t think that’s the cause – though I might be missing some knowledge about my protein – I will look more into it, but what made me suspicious – I had the same problem with 2 different proteins, so I believe that the cause is rather technical

I will try unstained marker too… I also used Kaleidoscope before – though when I worked in a different lab and I used gels from BioRad, (here we use Invitrogen precast gels), and I had no problems with it

Today we had a vendor show and I talked to BioRad rep – she said I might use a wrong gel for the proteins of this size: 120 – 160 kDa and 8-16% gel. I might try 7,5% or 10% gel to rule out this possibility, though I believe it is a right gel.  (I also planned to reprobe the gel for the smaller protein, later).

Thank you again, I will  keep you informed about the results, if you are interested

[mikew]

Hi,


Yes, it would be great to know the result of the Actin probe.
I am very curious!

Good luck!