Hi everybody. In attachment you will find my latest running of a human plasma 2DE. I have run MARS14 before and the collected proteome profile is shown. Can any body suggested how I can improve the gel? Where do I do wrong since I have not a good resolution? It seems that I have problem with focusing in first dimension. If that is the case what can I do to improve the focusing?
Eradat,
Yes, it seems you have problem with the first dimension. You can make some changes in the last step of focusing, may be via increasing the focusing time (Vhr). Also check with the tank buffer, equllibriation time; add DDT and idoacetamide freshly.
Hi,
I agree with Prashant. Some alterations in first dimension will certainly improve the protein separation. Changes like give enough time (two steps of total four hours) at low voltages (up to 500 volts), increasing the volt hours, keeping paper wicks, giving adequate time for reduction and alkylation (15 mins and 30 respectively). If you are leaving the IEF in lower voltage after the run you should refocus it with the maximum voltage it reached for at least half an hour.
Gud luck
Hi Kambiz,
More information is necessary to properly troubleshoot your results. Elution conditions for the MARS14 column (high salt, low pH, something else?), did you run eluate and retentate samples by simple SDS-PAGE to first evaluate the effectiveness of the pull-down by the column, were protein samples dialyzed or de-salted prior to IEF loading, amount of protein and volume loaded onto the IEF strip, passive or active rehydration, IEF system, pH gradient of strip, IEF running conditions, was the IEF strip overlaid with acrylamide or agarose, what steps did you conduct after IEF to prepare the strip for SDS-PAGE, SDS-PAGE conditions to properly interpret the 2D gel image, etc.
Dear Kent. It was a lot of points. However, I can tell you it looks very nice on the SDS-PAGE. I will put the picture of the 1D so you can see it.
Merci,
Kambiz
Yes, many points, and each one could have an effect on your observed result. Based on your question "could it be because of salt", you probably eluted proteins off the MARS column with increasing salt concentration, correct? If so, what is the salt concentration (or approximate concentration) of your eluted protein sample, and how much of this was mixed with IEF buffer? In other words, what is the final salt concentration of the mixture loaded for IEF?
Dear Kent
We have not desalt the sample before first dimension. We used a filter, but we have take the sample which is over the filter and run the gel. Do you think it is because of salt we get this picture?
Merci.
We have used the MARS columns with great success to deplete plasma and CSF. I assume it is the non-bound fraction you are interested in. If you are using the proprietary A buffer for dilution and loading, the unbound (low abundant) protein fraction is coming off in very high salt which is used to minimize non-specific interactions with the antibodies on the column. You must desalt and concentrate the protein before running the IEF. This can be accomplished by using desalting spin columns (i.e. Zeba) equilibrated with a volatile buffer like 20mM ammonium bicarb. The resulting flow through protein can then be dried down on a speed vac to obtain concentrated protein with no salt. Molecules
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