12 replies to this topic

[bonku]

Hi,
Last few weeks I am having a strange problem in my protein running SDS-PAGE gels. Whenever I am casting a new gel (any %, from 8-14% that I tested) the gel seems to be fine. But when I finish running the gel i notice some bubbles at the lower half of the gel (typically 1-2 cm from lower end). There is no air cavity (bubble)when I open the gel. I do not feel any undulation or uneven surface but do notice a change in uniform consistency and some bubble like areas and/or a curved line at the bottom of the gel. All the bands that pass that area get messed up. I changed all the reagents and now using fresh SDS, TEMED, 40% Acrylamide-bis (from Biorad) etc;  but the problem still there.
Sometimes I can see the problem at the lower part of the new gel (before running it).
Any suggestion in solving the problem is appreciated.

[K.B.]

1. Do you degas solutions before casting gel?
2. What time and conditions do you use for polymerisation?

[bonku]

1. No, I do not de-gas solutions.
2. I cast the gel during normal working hours. I am not sure what you mean by "conditions".  
Solutions and other polymerization conditions are the same as before, when the gels used to be OK.

K.B., on 01 December 2010 - 02:07 AM, said:

1. Do you degas solutions before casting gel?
2. What time and conditions do you use for polymerisation?

[K.B.]

Try degassing your gel solution before adding catalysts - see if that helps.

Speaking of conditions I had in mind things like temperature. Once I saw bubbles between gel and plate when person used temperature (IR heating lamp) to speed up polymerization and be able to run electrophoresis in an hour after casting. I usually let the resolving gel to polymerize overnight at room temperature.

[bonku]

Thanks Veteran. I'll try de-gassing.
I do cast gel about a couple of hours before I run it. It is polymerized under normal temp and light condition (white fluorescent light in the lab at room temp).  
I used to have flawless gels with the same procedure till few weeks ago (without any extra measure, like degassing)!

[K.B.]

bonku, on 01 December 2010 - 07:39 AM, said:

Thanks Veteran.

"Veteran" is just a status description based on post count. My nickname/login here is "K.B.".

[mdfenko]

are you pouring your gels cold? if so then you may have gasses trapped in the solution. make sure your gel solution is at room temperature before you pour (then you shouldn't require degassing).

[bonku]

mdfenko, on 01 December 2010 - 08:09 AM, said:

are you pouring your gels cold? if so then you may have gasses trapped in the solution. make sure your gel solution is at room temperature before you pour (then you shouldn't require degassing).

All the solutions I use are at room temp (except 40% acrylamide/bis solution which is stored at 4C).

[bonku]

I tried the de-gassing of all solutions. But it did not help.  

bonku, on 01 December 2010 - 07:39 AM, said:

Thanks Veteran. I'll try de-gassing.
I do cast gel about a couple of hours before I run it. It is polymerized under normal temp and light condition (white fluorescent light in the lab at room temp).  
I used to have flawless gels with the same procedure till few weeks ago (without any extra measure, like degassing)!

[mdfenko]

you only need to degas the gel mixture prior to the addition of aps (and temed, if you wish).

maybe your plates aren't as clean as they seem. very slight contamination can be a problem.

or maybe your plates have started to warp.

[bonku]

I clean my (already cleaned by soap and dist. water) plates with EtOH before pouring gel.
What do you refer by "wrap"?

mdfenko, on 07 December 2010 - 11:34 AM, said:

you only need to degas the gel mixture prior to the addition of aps (and temed, if you wish).

maybe your plates aren't as clean as they seem. very slight contamination can be a problem.

or maybe your plates have started to warp.

[knuf]

How are your combs looking? If they don't fit snugly (like if the end piece breaks off), then it can do some screwy things to your gel.

[mdfenko]

bonku, on 07 December 2010 - 11:52 AM, said:

I clean my (already cleaned by soap and dist. water) plates with EtOH before pouring gel.
What do you refer by "wrap"?

that's "warp". the plate can become less flat (bent) over time.