8 replies to this topic

[sale9yu]

Greetings everyone!
The proteins I use, CytC, neuramidase, histone and even MW marker run faster than the dye front. All solutions are made fresh and 15% gels are used. After adding the 2x loading buffer the samples are heated at 90C for 4 min.
Would you, please, be so kind to help me solve this problem. Thank you!

[Curtis]

I have detected Cyt C at 15kb on 15% gel. shouldn't be a problem. If you have problem with any protein running off (which I don't think so) then stop the gel sooner.

Edited by Curtis, 02 November 2010 - 09:48 AM.

[sale9yu]

Curtis, on 02 November 2010 - 09:47 AM, said:

I have detected Cyt C at 15kb on 15% gel. shouldn't be a problem. If you have problem with any protein running off (which I don't think so) then stop the gel sooner.

Thank you, Curtis. For this reason I do stop the run almost an inch above the edge. The problem is that everything including the MW marker outruns the dye front so something must be wrong.
Thanks for your post.

[mdfenko]

which tracking dye are you using?

phenol red runs faster than bromphenol blue.

what buffer system are you using (eg laemmli)?

[sale9yu]

mdfenko, on 04 November 2010 - 08:12 AM, said:

which tracking dye are you using?

phenol red runs faster than bromphenol blue.

what buffer system are you using (eg laemmli)?

Hi. Thank you for your post. I am using bromphenol blue and Laemmli buffer system. It looks like MW marker (biorad 161-0363)is running much faster than any other component.

[Curtis]

I wonder why, it shouldn't be. and how do you know they run faster? did you check them on western blot?

[sale9yu]

Curtis, on 04 November 2010 - 05:57 PM, said:

I wonder why, it shouldn't be. and how do you know they run faster? did you check them on western blot?

Hello,
Ever since I noticed the problem I use standards but I haven't done the WB. Also, MW marker is the one that runs the fastest. Out of 10 possible bands I see only 8. Thank you!

[mdfenko]

the pH of one or more of your buffers may be off (you can't properly check them in the presence of sds, maybe with a solid state electrode).

[shivasankari]

sale9yu, on 02 November 2010 - 08:49 AM, said:

Greetings everyone!
The proteins I use, CytC, neuramidase, histone and even MW marker run faster than the dye front. All solutions are made fresh and 15% gels are used. After adding the 2x loading buffer the samples are heated at 90C for 4 min.
Would you, please, be so kind to help me solve this problem. Thank you!

Hi,
  There might be an uneven distribution of the gel components as well! And sometimes when the dye is very old you can encounter this problem. Its nothing to worry about, just stop the gel soon and if you are very much worried about the marker, you can use a pre-stained one which will help you keep a track of the run.

Cheers

Shiva