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2D Image Primary Cell line - (Apr/19/2013 )

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My project is about proteomic study of human primary cell. Till now I do not have get good result from my 2D. I run 12 3-10 NL strips and I attached the last one but I didn't improve. Is there any expertise to help me. Thank you
Attached File

-virusproteomics-

you can download a useful handbook on 2d electrophoresis (as well as other useful handbooks) from ge healthcare life sciences.

-mdfenko-

mdfenko on Mon Apr 22 12:40:36 2013 said:


you can download a useful handbook on 2d electrophoresis (as well as other useful handbooks) from ge healthcare life sciences.
Thank you very much for this helpful link. Anthor challeng with my 2D is that always when I run strip at the + end of the strip there is whitish percipitate and I don't know what is that and after second dimension shows that this region is not focusing. I don't know how to solve it.

-virusproteomics-

are you using a narrow or wide range ief strip? if narrow, try a wide range to see if you can resolve that area.

the precipitate may be a component of the sample's buffer (edta?).

-mdfenko-

I'm using 3-10NL and 4-7 strips. In both cases I can see that area but I think the area after this whitish area also can not focus and I have smear area in second page. I can not solve this problem

-virusproteomics-

we need more information. in what buffer is your sample? how much do you load? how do you load?

have you tried linear 3-10?

-mdfenko-

usually I load 10-15 ug protein. I mix it with sample re hydration buffer to the final volume of 125-140 ul. I never tried linear one. always used non linear. can I get better result by linear?

-virusproteomics-

non-linear causes compression at the ends and may be the cause of the white precipitate and unfocussed region.

in what buffer is your sample prior to mixing with rehydration buffer?

i assume from your response that you rehydrate the ief strip with the sample (which is, of course, the right way to introduce sample to dry strips). do you let the sample absorb sufficiently? does the strip remain "drippy" wet?

-mdfenko-

Thank you very much for the reply. My sample is prepared in 1.5% Carrie ampholyte. then I use 0.5% in the sample rehydration step. I leave the strip to absorb sample for 30 min before I add the mineral oil. and consequently leave it for at least 12 hrs in the passive re-hydration I've attached my latest gel image for your perusal.
Attached File

-virusproteomics-

how do you lyse your cells?

i think that some buffer component is precipitating in the gel and clogging the pores (leading to the unfocussed region).

if not a buffer component then you'll want to precipitate the proteins and resuspend them in resuspension medium (you may have to dialyze after suspension depending on which precipitation method you use).

you may also benefit from using "destreak reagent" in your resuspension medium.

-mdfenko-
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