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There have been 10 items by polaris (Search limited from 02-April 19)


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#106785 Poll: Time and voltage for wet transfer to PVDF

Posted by polaris on 14 April 2011 - 07:44 AM in SDS-PAGE and Western Blotting

[/quote]
Hola, your conditions are adequates for transfer, so have you check polarity, and activate PVDF with methanol For me these are the causes to fail. Buena suerte
[/quote]


Polarity is correct, transfer goes from black to red side, gel is on black side membrane is on red side, PVDF is activated and rinsed...



#106773 Poll: Time and voltage for wet transfer to PVDF

Posted by polaris on 14 April 2011 - 07:06 AM in SDS-PAGE and Western Blotting

15 v
overnight.
0.1 % SDS

What size range is your protein/marker? Most proteins don't blow through PVDF.



Markers go from 14k to 97k.



#106696 Poll: Time and voltage for wet transfer to PVDF

Posted by polaris on 13 April 2011 - 01:28 PM in SDS-PAGE and Western Blotting

I'm trying to use tris/glycine/20%MeOH (Towbin's) with 0.1% SDS to wet transfer onto PVDF. I think I'm blowing right through the membrane; my markers are not on the membrane or the gel after 100V for 2hrs.

If you use Towbin's buffer and a wet transfer system to PVDF (mine is bioRad mini):

What is your:

Voltage:
Time:
SDS concentration:



#87522 What causes this offset band on RIPA gel?

Posted by polaris on 21 September 2010 - 07:23 AM in Protein and Proteomics

In lane 1 there should be a band at 68 and in lane 2 there should be a band at 67; these should be the two darkest bands on the whole gel. Reagents are about as fresh as they always are when the assay works... I rinse the wells with running buffer and then aspirate to clean them out before loading samples.

I'm thinking about the samples themselves. After immunoprecipitation the samples are eluted off of protein-A sepharose beads (in loading buffer with DTT) by heating in boiled water for 5 min., then they get frozen overnight. The samples are thawed at room temperature the next morning while the stacking gel is being prepared (resolving gel is prepared the afternoon before). When the gel is ready, we vortex and centrifuge the samples and then load.

I'm thinking that it wouldn't hurt to reheat, before spinning down and loading. Since I only see this problem with these two higher wt. proteins, could it be that they are more prone to forming some kind of precipitate or secondary structure at low temperatures?



#87440 What causes this offset band on RIPA gel?

Posted by polaris on 20 September 2010 - 02:07 PM in Protein and Proteomics

Thanks for the reply.

I do centrifuge prior to loading. I suppose I could do it longer and harder. Would your explanation fit the observation that this only seems to happen with the two proteins I'm interested in at around 68kd?



#87428 What causes this offset band on RIPA gel?

Posted by polaris on 20 September 2010 - 09:05 AM in Protein and Proteomics

This is an autoradiograph of a RIPA gel. The band at the arrow will sometimes show this odd stepped appearance, sometimes it's smeared and sometimes it's just not there. Also the standards in the far right lane are somewhat diffuse. Any experience with this condition?

This is SDS-PAGE with reduced samples (DTT)...

Attached Files




#74870 Methanol vs Isopropanol in fixing solution

Posted by polaris on 10 June 2010 - 08:46 AM in SDS-PAGE and Western Blotting

General question: What is the difference between using methanol/acetic acid versus isopropanol/acetic acid when fixing proteins in gels?



#71830 Reviving a hybridoma from LN2

Posted by polaris on 20 May 2010 - 07:27 AM in Immunology

Hi all,

We're about to revive some frozen hybridoma cells and the procedure recommends co-culturing with normal mouse spleen cells until the hybridoma cells can proliferate on their own. I'm having trouble finding a source of normal mouse spleen cells.

1. Is it necessary to co-culture?

2. Sources of normal mouse spleen cells?

3. Alternatively I think we can just use conditioned medium but the source I found (Sigma) has it back ordered until July. Any other conditioned medium sources?

Thanks!

-P



#71828 Preparation of peripheral blood mononuclear cells

Posted by polaris on 20 May 2010 - 07:08 AM in Cell Biology

You should be starting with a spin of the whole blood without any ficol. The RBCs will be at the bottom, then the buffy coat where your PBMCs are, then plasma on top. Remove plasma with a pipette; this can be clotted with CaCl if you want serum. Skim off the buffy coat and do ficol on that.



#70482 small proteins have run ahead of tracking dye

Posted by polaris on 12 May 2010 - 09:24 AM in SDS-PAGE and Western Blotting

Hi all.

I run an acrylamide gel electrophoresis for RIPA and have encountered a new problem after preparing some new reagents. The smaller mol. weight proteins have run off the gel as if I let it run long, but the tracking dye had only just reached the bottom of the gel. Tracking dye was one of the new reagents. Any other likely candidates? I run at constant power (amps) and the voltage started and finished a little lower than usual - usually starts at ~107v and runs up to ~390V after 4 hours, but this time 97v to 335v after 4.5 hrs. I also decided to make my stacking gel a little taller - usually 1cm from well bottom to top of resolving gel, this time 2cm, but I've run it this way before without the run-off problem.

Thanks for any advice...

-P




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