Make sure the electrode solutions were made at the correct concentrations.

Hi MDV,

In many cases, the issue of not reaching the set voltage is associated with too high ionic strength in the sample.

Albeit you have desalting your samples, did you purify your samples close to the efficacy specified by the 2D clean-up kit?

Have you tried changing the IEF paper wicks more frequently?

Another way to get around could involve this:

Pause the program, take the strip out, rinse briefly with double distilled water, blot away excess water, and resume the IEF. This could help remove more of the interfering ingredients

Hi Kay wong,

Thank you very much for the suggestions.

We didn't use any 2D cleaning kit,instead we have done chloroform:methanol precipitation to remove the unwanted materials from the protein sample. Is it because of the reason that I have added more ampholyte more than the desired concentration that might have interfered in reaching the desired voltage? The voltage is ramping but very slowly. Yes we do change the paper wicks frequently, like one wick at each electrode for each run.

We are using pH3-10 7cm IPG strips and the concentration that has to be added should be between 75-120ug as per Biorad protocols. But we have loaded extra sample (around 200ug) in the final 125ul of the rehydration buffer. Will the overloading of the samples could be a issue in getting the voltage???

Thanks,

Hi MDV,

The ampholytes were mainly used to facilitate the electrical conduction and as a pH buffer. If the pH range (of the amphoplytes) used is correct, I guess a little bit more should not be a concern.

If you need that much of a loading amount, slightly increasing the rehydration volume (e.g. 10-15%) may help improve focusing.

Have you used the protocol for 2D gel run before? I have not tried chloroform:methanol precipitation before on 2D gels. Instead, our lab (and many studies out there) uses 2D clean-up kits (similar to the TCA/Acetone method).

Hope this helps.

Hi Wong,

Thank you for the suggestions.

We are standardizing the 2D protocols. But this is the 8th strip that we are using just to standardize, but no results !!!!. I don't think methanol:chloroform shouldn't be a problem because its better than TCA/Acetone method in precipitating proteins as it removes unwanted salts,lipids etc from the protein sample. Any problem with the 2nd dimension?? we have used 10% gels for the separation followed by coomassie staining. It would be of great help if you could send me the detailed protocol from the sample preparation till the 2nd dimension, if you do have so.

Thanks,

Hi MDV,

I followed the manuals by common vendors (e.g. GE, Bio-rad), where you could freely download them elsewhere.

I guess the 2nd dimension would be more straight-forward. The manuals from the vendors also have trouble-shooting guides in them, which would be of more help.

Good luck :)

Thank you Wong !!!

Dear Ivana,

Thanks for the suggestion and we have never tried that option. Will try to do it.

Also make sure the samples are well sonicated to fragment genomic DNA which can really slow your focussing down and produce poor results. I then precipitate using the pierce proteoextract precipitation kit which is a modified acetone precipitation method to desalt.

Hi Susana,

That was indeed a great help because  we we confused weather to go with Chloroform:methanol or TCA:acetone for precipitating proteins. May be we will try this and hope will be getting good results.

Thanks-MDV

sometimes improper protein solubulization also leads to the similar problem. 

make sure proteins are completely solubulized with proper buffers.

Hi, MDV,

Susana's suggestion is OK. If your IEF instrument is at good state, the un-desired voltage is possibly induced by salt-content protein sample, or salt-contaminated protein sample, or high-concentration carrier ampholytes added, or over-loading protein sample.  The details of relevant mechanism was revealed ( Talanta, 2013, 111, 20-27).

Hope the discussion is  useful for the run of IEF.

Chengxi

sometimes I have the same problem. Generally, all of the people writing above are right. Cheng-Xi has summed up the cruicial points. I used to chnge paper wicks but, what is more important, I use 2D cleanup kit form Bio-Rad. I rather do not precipitate my proteins (I don't have to) but for me it is interesting to dialize them. I was always afraid of that, it seems to be time consuming. Thank you for briging up this issue and good luck!