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2D Image Primary Cell line

Primary cell line proteo

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13 replies to this topic

#1 virusproteomics

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Posted 19 April 2013 - 10:27 PM

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

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#2 mdfenko

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Posted 22 April 2013 - 04:40 AM

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

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Posted 08 May 2013 - 08:40 AM

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.

#4 mdfenko

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Posted 09 May 2013 - 04:46 AM

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?).
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#5 virusproteomics

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Posted 15 May 2013 - 09:48 AM

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

#6 mdfenko

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Posted 16 May 2013 - 04:01 AM

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?
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#7 virusproteomics

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Posted 16 May 2013 - 06:49 AM

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?

#8 mdfenko

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Posted 16 May 2013 - 10:52 AM

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?
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#9 virusproteomics

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Posted 16 May 2013 - 08:46 PM

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.

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#10 mdfenko

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Posted 17 May 2013 - 11:27 AM

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.
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#11 virusproteomics

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Posted 20 May 2013 - 08:32 AM

the only thing that I'm suspected about my sample is that I'm using primary cell line from trachea origin in the human lung. it is epithelial cell and most probably produces strong mucous. When I'm centrifuging my cells it appears as a strong clump which is hard to re-suspend. I assume that the mucous is making this problem in strip running and focusing. I'm not sure it is right or not. If the mucous is the problem I don't know how to treat my sample.

#12 mdfenko

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Posted 21 May 2013 - 03:31 AM

can you wash the mucous away from the cells before you lyse them?
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#13 virusproteomics

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Posted 21 May 2013 - 05:17 AM

I m washing it with PBS but I think it may can not remove it.

#14 mdfenko

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Posted 22 May 2013 - 04:53 AM

you may need to add a little non-ionic detergent (not enough to disrupt the cell membrane).
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