Immunoblotting

Immunologic method used for detecting or quantifying immunoreactive substances. The substance is identified by first immobilizing it by blotting... | Contact experts in Immunoblotting to get answers

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Questions related to Immunoblotting

Has anyone analyzed porcine Cytochrome C via Western Blot?

We are looking for a primary antibody against cytochrome c known to work in pigs. Thank you!

04 April 2015 8,079 3 View

Why couldn't the antibody recognize the endogenous protein?

I want to test the protein expression of a protein around 130kD but I could not detect it. When I over-expressed the protein, I can detect the protein but not the endogenous protein. I tested...

04 April 2015 2,752 17 View

Which autophagy related proteins have a short life time in primary human cells?

Many autophagy related proteins, like Beclin1, ATG7, etc. have a certain half life time. Which proteins are degraded at a relative high speed (so within 24 hours)? Does anyone tested a certain...

04 April 2015 3,648 4 View

Is it possible to have ghost bands on immunoblot because polyphenols have been transferred with the proteins and block antibody binding?

Hello scientific world! I would appreciate your insight/experiences with the following: Has anyone else observed that polyphenols that have been complexed with proteins can (after SDS-PAGE) be...

04 April 2015 3,118 5 View

Which side of PVDF membrane after transfer should I use for probing and for imaging?

Western blot. Transfer using PVDF or Nitrocellulose. Is it recommended to probe ab on facing side (the side that is facing gel)? Or it doesn't matter at all.

04 April 2015 8,315 2 View

Where can I purchase antibodies for a western blot assay?

My research team have been conducting protein profile studies for heat shock proteins but we need to be able to quantify the induced proteins.

04 April 2015 8,242 7 View

What are the factors that affect the electrical current and voltage during protein blotting (western blot)?

I have one gel and I set the ampere limit to 0.11, but the actual amber value unstable (0.16 to 0.21) and the voltage is about 22. So why the actual ampere is higher than the adjusted one.

04 April 2015 6,242 2 View

Did someone ever stripped a lectin blot on PVDF membrane?

I am planning western blots using different lectins to test the glycosylation of some samples. As the material is often available in limited quantity (e.g. patient material) it would be great to...

04 April 2015 6,353 7 View

Can anyone recommend a good anti beta arrestin antibody for immunoflurescence use?

I already tried a Santa Cruz one but it doesn't work very good.

04 April 2015 8,727 2 View

Does boiling cell pellet in PAGE SDS-loading buffer efficiently release membrane-bound proteins?

My immunoblotting failed to detect a protein of interest and I'm using the M-PER™ Mammalian Protein Extraction Reagent, which could efficiently extract both cytoplasmic and nuclear proteins...

04 April 2015 8,942 5 View

Can anyone suggest how I can overcome the faint bands in western blotting?

I am using DAB staining to detect signal for protein expression, but now a days I am getting faint bands? Even increased the protein concentration, fresh antibody dilution, reduced blocking steps...

04 April 2015 8,934 5 View

Which commercially available anti-His antibody has the best performance for immunoblot/Western analyses of 6His-tagged proteins?

I have tried a few primary antibodies to detect His-tagged proteins on westerns, and been disappointed with the low signal intensity compared to other primary antibodies used in my lab. I would...

03 March 2015 1,000 9 View

Is sonication a preferable method in preparing protein lysate for immuno blot?

What kind of differences can be seen in the sample with or without sonication?

03 March 2015 9,849 4 View

Does anyone has experience of separating Dopamine 2 long and Short form using SDS-PAGE?

Does anyone has experience of separating D2long and short form using SDS-Page? I have run the mouse brain tissue sample on SDS-page at 165 v for approximately 3 hrs to separate D2long and short...

03 March 2015 1,944 2 View

Could you explain this multiband western blot with p-H2AX?

Hello all, We are interesting in studying p-H2AX in lymphocytes. At the same time we see some other proteins, the bottom of the membrane is cut and incubated for p-H2AX (cell signalling,...

03 March 2015 8,936 6 View

Has anyone been using "1-Step™ NBT/BCIP Substrate Solution" for developing western blots?

Our group is looking at moving away from traditional western blot methods as there are many more time efficient options now available. I have seen an impressive demonstration of using a few ml of...

20 February 2015 1,177 3 View

Has anyone made electrotransference in Transblot Turbo equipment of BioRad using acrylamide gels 1.5mm?

Hi, we tried to transfer proteins from an acrylamide gel 1.5mm made with our recipe to a PVDF membrane of BioRad in the Transblot Turbo of BioRad, but the majority of protein rested in the gel....

10 February 2015 6,220 4 View

Has anyone been using "1-Step™ NBT/BCIP Substrate Solution" for developing western blots?

Our group is looking at moving away from traditional western blot methods as there are many more time efficient options now available. I have seen an impressive demonstration of using a few ml of...

02 February 2015 6,571 3 View

ChIP-Western : what is the advantage?

Hi, I recently came across 2 papers mentioning ChIP-Western :typical ChIP for protein A followed by elution of proteins (as opposed to DNA) and Immunoblotting for protein B. This seems to be a way...

02 February 2015 9,009 0 View

What are proper housekeeping genes in the plasma membrane fraction for western blotting?

I studied the regulation of Caveolin-1. I used coomassie staining of PVDF membrane as loading control for western blotting of membrane fraction (isolated by differentiational centrifugation). But...

02 February 2015 2,235 20 View

Why do I get a double band when performing western blot analysis of the PAH protein in Hek 293 cells?

We found a band at the expected molecular weight and another 1-2 kD lower with almost the same intensity. We are using an anti-DYK antibody, and our expression plasmid is pcDNA3.1+. The DYk flag...

02 February 2015 7,421 3 View

Has anybody ever worked with RGS2 antibody for Western blot?

Do you have any suggestions for the antibody/ies that really works? It shouldn't make bands on RGS2 -/-

02 February 2015 2,821 1 View

Is it possible to do a second ECL-based detection, with freshly made ECL, on a nitrocellulose membrane 1 week after you first detection?

I succesfully detected my protein of interest, but now, 1 week later, I want to do the detection again. Will I be able to detect my protein of interest by simply incubating it in freshly made ECL,...

02 February 2015 8,499 3 View

I am working on optimization of native PAGE,I am unable to get my protein of interest after staining,what could be the reason?

I am working on optimization of native PAGE but I am unable to get my protein of interest after staining. I have read several protocols but I am still confused. I am also worried about the loading...

02 February 2015 3,784 23 View

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