7 replies to this topic

[olga_m]

Hi Guys!

I have a feeling that the protein marker (i use Fermentas) may not always represent the actual mol weight (kDa) of the protein. Many times i get my band/protein of interest slightly (but significantly) above or below the marker protein of the same MW. Has anyone noticed this kind of issue?

Thank u very much
Olga

[HomeBrew]

Pre-stained markers are not good for molecular weight estimation -- they're most useful for monitoring the progress of your gel run and for judging western transfer efficiency, but because they're tagged with a dye molecule, their migration through a gel is greatly affected by the conditions of the run -- pH, the buffer system used, etc.

See, for example, the difference in apparent molecular weight of the same Fermentas pre-stained marker run under differing conditions here.

[olga_m]

Pre-stained markers are not good for molecular weight estimation -- they're most useful for monitoring the progress of your gel run and for judging western transfer efficiency, but because they're tagged with a dye molecule, their migration through a gel is greatly affected by the conditions of the run -- pH, the buffer system used, etc.

See, for example, the difference in apparent molecular weight of the same Fermentas pre-stained marker run under differing conditions here.

Thank u very much for replying so quickly to my post! Could you suggest me a different protein marker you think would be more effective?

[HomeBrew]

Are you trying to size a protein band by staining the gel with a general purpose stain like Coomassie? If so, an unstained molecular weight marker like this one should do the trick...

If you're trying to size your band after detection on western blot, it's a bit trickier... Which are you doing?

[olga_m]

Are you trying to size a protein band by staining the gel with a general purpose stain like Coomassie? If so, an unstained molecular weight marker like this one should do the trick...

If you're trying to size your band after detection on western blot, it's a bit trickier... Which are you doing?

i am trying to detect a band of 120kDa and I take a few bands close to this molecular weight. My marker is 120kDa too. One of the bands seems to have the responses i expect (activation/inactivation) but it is a bit higher than the respective marker. The band that appears exactly at 120kDa doesn't seem to change significantly. The situation turned to be more complicated when I ran a western blot for a different protein of the same molecular weight and the band appeared lower than the 120kDa marker this time. In both cases I used the same aliquot of fermentas protein ladder.
I am so confused........!!!

[HomeBrew]

So, you're detecting your protein by western blot? What antibody are you using to detect your protein? What is the nature of your sample -- is it a whole cell lysate or purified protein?

[olga_m]

So, you're detecting your protein by western blot? What antibody are you using to detect your protein? What is the nature of your sample -- is it a whole cell lysate or purified protein?

Yes, I'm detecting my protein by western blot. I use polyclonal first antibody against one protein and monoclonal against the other one. The second antibodies are HRP-conjugated, anti-rabbit and anti-mouse respectively. My samples are total cell lysates.

[HomeBrew]

If accurate sizing on the western is needed, you could try a biotin-labeled ladder compatible with your HRP detection, like this one.