2D troubleshoot

[admin]

Hello Aaina, dont worry the first step is the hardest so we can fix the second step eventually.

The second gel dimension issue could be due to several reasons.

- Did you dialyze your human plasma at all with buffer? Could be a salt issue.
- You could have had Insufficient volume of buffer in upper or lower reservoir for the second dimension.
- Buffer for second dimension was not prepared properly
- Did you use fresh buffers for the second dimension?
- Could be a gel problem - What percentage is the gel?


Aaina is this your first time running the apparatus? If so try to get someone to help with its setup / buffer prep and running.
Get back to us please,
and if you do re-run let us know what happens
good luck

[deepee]

How did you lyse the cells that you collected from your plasma sample and how do you solubilise the protein before rehydration? How much protein did you run on your strips and were they 17cm pH 3-10 strips?
I'm sorry about this but the more detail you can give the more likely someone on the forum will be able to help.
There are many stages to running succesful 2D gels and each of them have many variables, it'd be really helpful to know more about the solubilisation reagents, focusing times etc.
What is your tissue sample? I've only used brain and epithelial cells from swabs but methods are reasonably transferable so I should be able to point you in the right direction.
Thanks,
deepee

[dky9829]

the IEF didnot work well! Do you use Ampholine ?

[mlhdopamine]

Hi
by the looks of your picture it seems I am having a similar problem with my 2d gels. As far as the second dimension, that should be easy to trouble shoot. Simply rehydrate your gel strip with sample>>>DO NOT IEF>>> and run the gelstrip for the last and vertical dimension. If you get what would look like a 1d western blot with one big lane then the second dimension should be ok.

However, i think deepee touched on our problem which could be properly solubilizing the protein sample.
Here is my brief protocol.
50 ug of bovine whole retina lysate (celLytic MT). 40ul sample and 80ul rehydration buffer.
Rehydration buffer =
a. 2.6 gm Urea
b. 1ml Triton-X
c. 100µl IPG buffer pH 3-10NL
d. 50µl of 2M DTT
e. Bring to 5ml with water and vortex thoroughly
f. Add a pinch of Bromophenol Blue and re-vortex

I rehydrate the total 120ul to a 7cm gelstrip overnight with a layer of mineral oil and proceed to IEF.
1. 250V for 15 minutes
2. 4000V (end voltage) 20,000vh at rapid ramping
3. 500V hold step.

After that everything is the same with the equilibration buffers I and II ( DTT and Iodoacetamide, respectively)

Anyway my 2d gels turn out pretty much the same as Aaina's so if anyone has any ideas please email me [Only registered users see links. ].

[deepee]

Hi mlhdopamine,
are you really using 20% TritonX in your rehydration solution? Is this in your solubilisation solution too? I think you should drop that by factor of ten to 2%, 4% at the most. Your Urea solution is at a high concentration too, not worryingly high but at 8.6M it's higher than most use it at, you shouldn't have a problem with the solubilisation of non-hydrophobic (membrane) proteins at this level, especially if you add in up to 3M thiourea too.
You can add up to 4-5 times that amount of DTT (you're using 20mM) if you think oxidation is a problem, I use 1% DTT (~65mM).
I wouldn't vortex it too fast either, your detergent will probably froth up, this is a minor point though.
Other than that, the only things I'd suggest are allowing your sample to rehydrate into the strip in the absence of mineral oil for an hour or so before you add that over, stepping up your focusing protocol more gradually (e.g. 250V 15mins, 500V 30mins, 1000V 1hr, 4000V to 20KVhrs; all rapid ramping), include 5mM DTT in your cathodic end wick, make up your agarose overlay in 2xSDS running buffer and make sure it isn't too hot (but obviously still molten) when you overlay it, and finally, start your 2nd dimension run at a low voltage for the first 15-30mins, then step that up too, maybe in 2-steps to your final voltage.
I've never used the samples you're dealing with, and I use different components (thiourea, CHAPS etc.) in my buffers, so I can't really comment in too much detail about the specifics of your problems, but I think you'll easily be able to improve on the results you've been achieving so far. Do you know if the samples are likely to have a high proportion of basic or hydrophobic proteins? If so you've got a hard job on your hands. I avoid those kind of samples like the plague and leave them to LC-MS people to deal with.
Good luck anyway, let me know how you get on.

[dky9829]

the hot SDS method is better for plasma sample. i think your isoelectric focusing not enough! 7cm 5000v 20000vh? how much did IPGbuffer you use?

[zafar1231]

Dear Aaina
U can do same general sds page for that. u need not to worry for plasma protein . it will give u good result if ur strip was fully focused.