Western Blot

Identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of... | Contact experts in Western Blot to get answers

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Questions related to Western Blot

Why do I have background when I detect the nitrocellulose membrane?

Dear all, I performed Western blot on plant material by using specific antibody raised against rabbit. When I try to detect the proteins, I see background from the nitrocellulose membrane (see...

11 November 2019 5,397 4 View

4-HNE Antibody stains Mili-Q H2O. Where is the problem?

Dear colleagues, We are having an issue with the Anti-4-Hydroxynonenal Antibody (AB5605 from Merck). We've been using this antibody for the Slot Blot technique, but it seems to react to just H2O...

11 November 2019 5,157 2 View

Why my PBMC lysate has negative value but it can blot and has specific band appear??

I would like to study proteins expression but now i am in try condition process. I used PBMC for cell lysate preparation and the protein concentration always had negative value (-1 to -2). Anyway...

11 November 2019 1,766 3 View

Anyone seen Western blots look like this?

This has been a problem with my Westerns lately, I'm pretty sure it's not a transfer issue, but it looks like the secondary antibody is being scraped off somehow? I'm not sure how this could be...

11 November 2019 6,673 4 View

Western Blotting of very high MW proteins - how to?

Hello everyone, I'm constantly struggling with one issue. I have to detect dystrophin (427 kDa) on western blots. I use 4-15% TGX-PAAG and have tried dozens of different transfer protocols....

11 November 2019 1,230 2 View

Can anyone help me with the detection of amyloid beta by western blot?

Can anyone help me with a working protocol for amyloid beta detection by western blot? I have extracted the whole proteins from the cells. For the Western blot I used a Precast gel 4-20% (in SDS...

11 November 2019 3,113 4 View

How to isolate proteins out of PEGDA hydrogel?

After extracting proteins out of PEGDA with Ripa and sonication i had quite good protein concentrations, but there were no bands in my Western Blot. Ponceau and Coomassie showed that there was...

11 November 2019 5,356 1 View

How to test a protein in western blot from cell lysates without overexpression?

I want to show one protein (~70kDa) in one cancer cell line lysates using western blot, but no transient overexpression. Is that possible?? And I have use GENEVESTIGATOR to find out that the...

11 November 2019 6,263 3 View

How to detect gamma H2AX and cleaved caspase 3 signal for WB?

I am trying to detect any differences in DNA damage/apoptosis related proteins by western blot between wild type and disease cells. I ran an initial WB and saw no bands for either protein and am...

11 November 2019 5,707 4 View

How to preserve plasma samples for further protein extraction?

I’m planing on performing protein extraction from blood Plasma. What is the best way to preserve the sample before the extraction? shoud I freeze it in -20 as blood in ETDA tube? Or separate the...

09 November 2019 8,381 2 View

What is the best and the cheapest protein detection method ?

I will search for the existence of a protein in 400 samples. I need the best and the cheapest technique that makes the detection of my protein's quantification possible. My budget is limited and...

06 November 2019 4,154 4 View

What will be the perfect markers to differentiate Neural Stem cells from differentiated Neurons?

I've been working on differentiating Rat fetal Neural Stem cells into Neurons with a drug of interest. The Problem here for me is I have used various Neuronal markers like MAP2, TUJ1, NFM to...

16 October 2019 7,379 5 View

How can I know my sample (cell medium) are too condense of cytokine before performing ELISA?

1. When performing ELISA, we have 2 different panels: standard and sample. In western blot, we do BSA assay at first to quantify protein concentration, so if the protein concentration is too much,...

11 October 2019 3,985 1 View

Which and for how much time kan I keep Western Blotting solutions?

I'm starting a series of WB experiments in my lab, and it'll be the first time that I do this technique. I read a bunch of protocols and realized that is required significative number of different...

10 October 2019 1,649 4 View

Think that the reason is sulfhydration of the protein, how can confirm that?

Hello all, I have noticed a shifting to low molecular weight band of protein by Western blotting after incubation with H2S donor (presence vs absence of H2S), I think that the reason is...

10 October 2019 872 1 View

How can I know my sample (cell medium) are too condense of cytokine before performing ELISA?

1. When performing ELISA, we have 2 different panels: standard and sample. In western blot, we do BSA assay at first to quantify protein concentration, so if the protein concentration is too much,...

10 October 2019 396 1 View

Does anyone have a good protocol for separating mitochondrial, cytoplasmic and nuclear fractions from Arabidospsis?

I have a protein which might be dual localized in the mitochondria and in the cytoplasm. Therefore, I have to get them separated and check on the Western blot in which of these fractions I see my...

10 October 2019 9,567 1 View

If the lysates I have prepared for Western blot are frozen at -20°C before measuring protein concentrations, will the protein concentrations change?

I would like to measure the protein concentrations of the lysates I prepared for the Western blot method at the same time. How long can I wait for these lysates to remain stable?

10 October 2019 10,098 6 View

Detecting PD-L1 expression in cells ? Western blot or Flow cytometry?

Does anyone know what the best way to quantify PD-L1 expression is? Following treatment, I see a massive PD-L1 upregulation in mRNA level using qPCR and but only a partial upregulation using flow...

10 October 2019 3,769 3 View

Can mercaptoethanol speed up protein migration in SDS-PAGE?

We have done zymography and Western blot from the same samples. In zymography the detected band is at the size of 90kDa, but in Western blot at 70kDa. At both methods samples are in SDS-loading...

10 October 2019 1,721 7 View

How do you best store isolated exosomes?

I am isolating exosomes from the SBI Exoquick TC Ultra kit. I'm going to run some right away on western blots, but was hoping to store some for a while to add onto gelatin matrices for imaging....

10 October 2019 6,729 2 View

Hiv1 integrase western blot detection?

Hello everyone, I am working on a project for hiv 1 integrase. I have performed cloning and according my seq results it was successful. It has been few weeks I’m trying to detect my protein on...

10 October 2019 4,146 3 View

How can I fix my western blot membrane ?

I'm a beginner at western blotting, and I have hight background, However I don't know whether it comes from the primary or the secondary antibody. and I thought I could start over and incubate...

10 October 2019 5,224 5 View

Protocol for whole heart tissue lysis for Westerns?

Hello, I am troubleshooting a western protocol using whole heart homogenates but I seem to be getting consistent smearing at the lower band range of my gel which is also where I want to see my...

10 October 2019 1,634 4 View

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