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yoshimi79's Content

There have been 13 items by yoshimi79 (Search limited from 19-January 19)


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#73842 western exposure one part of band darker

Posted by yoshimi79 on 03 June 2010 - 08:21 AM in SDS-PAGE and Western Blotting

I generally mix the substrate around on the blot and then leave it for five minutes.

Do you have a preference for face up or face down in the substrate?



#72051 western exposure one part of band darker

Posted by yoshimi79 on 21 May 2010 - 12:12 PM in SDS-PAGE and Western Blotting

I see a similar problem and I'm not sure the cause. It seems like it could be from the transfer, but it's not always in the same place. Also it's only usually seen in highly expressed proteins.
See attached picture.

Attached Files




#69147 Phospho-proteins

Posted by yoshimi79 on 03 May 2010 - 07:33 AM in Protein and Proteomics

The same sample can give variable results between runs. Nothing extreme, but the error seems to be larger with the phospho proteins. I don't always normalize the sample concentrations before loading...



#68877 Phospho-proteins

Posted by yoshimi79 on 30 April 2010 - 08:17 AM in Protein and Proteomics

Does anyone have any tricks for Western blotting for Phospho-proteins? I'm looking at several different phosphorylated proteins in tissue samples and seeing a lot of variability.
Sample prep, Buffers, Transfer, freeze/thaw, boiling etc etc
I use protease phosphatase inhibitors in the homenization/lysis buffer, TBST and water to wash, and milk to block. Background is not an issue. Just highly variable signal



#65954 kinase activity

Posted by yoshimi79 on 08 April 2010 - 01:41 PM in Protein and Proteomics

Does anyone know how active kinases are at 4degrees?



#65892 OKADAIC ACID for protein prep

Posted by yoshimi79 on 08 April 2010 - 08:47 AM in Protein and Proteomics

Has anyone used okadaic acid as a phosphatase inhibitor when prepping protein from cell or tissue homogenates (for use in westerns)? I have a few questions

1. What concentration is necessary for PP1/2 inhibition? I've seen it used at multiple concentrations...

2. Is it possible to activate some kinase activity (it's been published that okadaic acid can activate PKC activity) in homogenized tissue that could confound the data. I keep my samples on ice and add 1mM EDTA, but I'm afraid that if phosphatases are inhibited without proper inhibition of kinases, the phosphorylated protein signal could be inflated.

3. How worried should I be about working with okadaic acid? It has a rather daunting MSDS....Any specific recommendations on handling?

Thanks.
Cheers.



#57056 immunoprecipitation lysis buffer

Posted by yoshimi79 on 29 January 2010 - 09:10 AM in Protein and Proteomics

I'm trying to measure differences in activity between genetically different mouse lines, so I need it pulled out of tissue lysate.
Thanks for all your advice though.
I was just worried about pulling down other proteins that may complex with my kinase that could produce nonspecific activity in the kinase assay. The assay is an ADP glo assay which measures the level of ADP remaining after incubation of kinase+ATP+substrate. I wanted to minimize the possibility that nonspecific substrates and kinases would included in my samples.
I really appreciate all your thoughts! Thank you!



#56684 immunoprecipitation lysis buffer

Posted by yoshimi79 on 26 January 2010 - 11:30 AM in Protein and Proteomics

1% SDS won't denature the protein?



#56198 DAB & DAPI together for ihc?

Posted by yoshimi79 on 22 January 2010 - 08:37 AM in Immunology

Yes, but i figured it would be easy enough to turn the fluorescent light on and off and overlay on another picture...



#56197 immunoprecipitation lysis buffer

Posted by yoshimi79 on 22 January 2010 - 08:35 AM in Protein and Proteomics

Does anyone know what homogenization/lysis buffer I can use to solublize the protein and disrupt protein-protein bonds (ie. I do not want to pull down complexes) but still maintain protein structure integrity and activity. I need to pull down a kinase and assay it's activity independent of other proteins.
Thanks.



#56092 DAB & DAPI together for ihc?

Posted by yoshimi79 on 21 January 2010 - 12:53 PM in Immunology

Does anyone know if you can double stain tissue (brain) using DAB to visualize the antibody stain and DAPI to visualize the nuclei? Only because we already have DAPI available in house...



#54889 Na/K ATPase as marker for plasma membrane?

Posted by yoshimi79 on 12 January 2010 - 07:59 AM in Protein and Proteomics

I'm fractionating brain tissue to look at nuclear pools of a particular protein. I want to be sure my fractions are clean, specifically rid of plasma membrane bound protein pools which may cloud small changes in the nuclear pool. Do you know if Na/K ATPase will serve as a good marker to detect contamination of the nuclear fraction? I'm not sure whether I would expect ATPase from the nuclear membrane to be present...



#54286 brain tissue fractionation (cytosolic and nuclear) protocol needed

Posted by yoshimi79 on 07 January 2010 - 09:29 AM in SDS-PAGE and Western Blotting

Does anyone have a good protocol for preparation of nuclear and cytosolic proteins from mouse brain tissue? I'm looking at a protein present in high levels at the membrane, so I'm especially interested in the purity of the nuclear fraction (ie. membrane protein contaminating the nuclear fraction).
Thanks




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