7 replies to this topic

[moland]

I forgot to put ice block in transfer buffer today, but the pres-stain standarded on the membrane looked just fine. Will it have any effect on my the protein transfer?

Thank you!

Edited by moland, 30 November 2012 - 07:00 PM.

[John Forsberg]

There's a possibility that your gel will warp if it heats up significantly during transfer, which can distort your protein bands.  It really depends on how hot your transfer buffer gets.  I usually transfer at about 300 mA for 90 minutes, and I never use an ice block because it never gets that hot in the chamber.

Back when we ran transfers at 1 A, I always ran those transfers in the cold room with an ice block though.  Your markers coming out fine suggests to me that your gel didn't heat up excessively.

[moland]

John Forsberg, on 30 November 2012 - 05:28 PM, said:

There's a possibility that your gel will warp if it heats up significantly during transfer, which can distort your protein bands.  It really depends on how hot your transfer buffer gets.  I usually transfer at about 300 mA for 90 minutes, and I never use an ice block because it never gets that hot in the chamber.

Back when we ran transfers at 1 A, I always ran those transfers in the cold room with an ice block though.  Your markers coming out fine suggests to me that your gel didn't heat up excessively.

Thank you! I run them at 100V for 1hr.

[John Forsberg]

In our system, 100V would probably give an amperage of about 700-800 mA, so it might still be getting quite warm (if I set constant voltage instead of amperage, it's usually ~40V).  Are you transferring in a cold room or fridge or at room temp.?  I didn't meant to make it sound like everything must be fine if the markers are okay, just that it's encouraging.  The only way to tell if any parts of your gel distorted is to probe with your antibodies (or you could do a Ponceau S stain for total protein on your membrane and look for lane/band distortions there, wash off the stain, then probe).

[moland]

John Forsberg, on 30 November 2012 - 08:19 PM, said:

In our system, 100V would probably give an amperage of about 700-800 mA, so it might still be getting quite warm (if I set constant voltage instead of amperage, it's usually ~40V).  Are you transferring in a cold room or fridge or at room temp.?  I didn't meant to make it sound like everything must be fine if the markers are okay, just that it's encouraging.  The only way to tell if any parts of your gel distorted is to probe with your antibodies (or you could do a Ponceau S stain for total protein on your membrane and look for lane/band distortions there, wash off the stain, then probe).

I probed the membrane today, and I got the specific bands i want.There is no distortion of lanes. However, my wild-type band is also shown in all my mut and non-transfected lanes, but not in the empty lane.  My mutant band is only shown in my mutant lanes. Can this be caused by the transfer at high-temp (because i don't think i contaminate my samples....)?

Thank you!

[John Forsberg]

What's your experimental setup?  Is this cells transfected with tagged wt and mutant proteins, then probing for the tag?  Or are you probing for an antibody specific to your protein (in which case the cells may produce wild-type protein)?

[moland]

It is tagged wt and mutant proteins, and I probe for the tag.

[John Forsberg]

And a band shows up in your untransfected lane?  Have you probed untransfected lysates from those cells before with that antibody and not seen a band at that size?  Usually even spillover will give a much fainter band in the lane it spills into.