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

What's the concentration recommend for recombinant protein use in western blotting?

I'm currently working having problem with the concentration for recombinant protein use in western blot, the one been used is Recombinant Human CD19 Fc Chimera Protein-rndsystem which does not...

25 February 2021 8,758 3 View

Has anyone experienced a double band GAPDH in Western Blot?

I am using my GAPDH as a housekeeping protein for my Western Blot. For all the blots I have, my GAPDH was coming out as two bands. Therefore, I am using both bands for my analysis. Has anyone...

12 February 2021 6,673 3 View

Western Blot Total Protein Transfer?

Im new to Western Blots. Im currently working under the analysis part in Image lab. I watched a couple of videos on how to use it and it says to upload two images (Total Protein Transfer and ECL)....

04 February 2021 2,138 3 View

Does Anyone Know of a Way to Detect Melatonin that is NOT HPLC, RIA or Mass Spec?

I tried to do a Slot Blot and was not successful and melatonin is only .23 Kda so it would probably run off a western blot. Has anyone had success with other methods?

24 January 2021 1,585 3 View

How to see the expression of 6kda protein?

Dear all, I am now investigating the expression of small size protein (6 kda). However, the expression of the protein cannot be shown clearly after performing western blot. Therefore, are there...

14 January 2021 3,324 3 View

Why are my western blot bands are think and faint?

Hi, my western blot bands are appearing at the correct molecular weight but are very thin and faint, making it difficult to distinguish difference in expression for shrna samples. Conditions...

05 January 2021 9,379 4 View

Minimal protein concentration from BCA can be detected using western blot?

hello, i extracted protein from PBMC's and after applying BCA assay on 2 samples the protein concentration was 400 ug/ml which means 0.4 ug/ul so does western blot work with these quantities? any...

25 December 2020 6,754 3 View

Is it possible that the phosphorylated proteins mobilize faster than the non-phosphorylated ones?

The band downshifts on Western-blot, after kinase assay treatment.

24 December 2020 8,135 3 View

How to optimize voltage and ampere for transfer of protein to PVDF?

Hello, I looked for a principle for the transfer procedure. I know that it depends on your instrument, buffer, Mw of proteins and the size of paper. I prefer to use constant ampere due to the...

22 December 2020 7,608 3 View

Unspecific band on western blot, what could it be?

I have a problem with unspecific bands on my blot. I have HEK cells transfected with a GFP construct (lane1), mCherry construct (lane2) and RFP construct (lane3). I collect the cells in PBS, do...

18 December 2020 9,411 3 View

Why do I get weird histone bands with my Hep G2 cells??

I'm performing standard histone extraction (abcam protocol) and western blot essay on my HepG2 cells but the band's that I get don't seem to have the classic histone shape. Any clue guys?

15 December 2020 1,833 3 View

How to avoid non specific bands in western-blots..?

I am working on autophagy.. trying to check the release of free GFP after autophagy by western blots.. but i am getting non specific bands.. how to avoid them and develop clean and neat blots ....?

15 December 2020 7,527 8 View

Blue native page transfer problem?

Hello, I am writing this hoping someone can help me. I am just starting out with BN-PAGE and I have a transfer problem. It seems each time I transfer (2 membranes at a time) in a BioRad wet...

10 December 2020 1,211 3 View

What is the difference between actin and loading control in normalization of western blot???

Regarding normalization of western blot analysis, I have found different strategies people follow but I couldn't understand the principle and which one is the accurate, 1- some refer to loading...

05 December 2020 5,894 8 View

Why is hard to produce antibodies that are specific to a protein?

Some antibodies reveal nonspecific bands when a western blot is performed. Why is hard to find a good antibody for a protein? Antibodies produced using as the immunogen a synthetic peptide or a...

04 December 2020 380 3 View

If we forget to add mercaptoethanol, can we correct the samples somehow?

Hi everyone, Can someone help me with a western blotting troubleshooting? So during sample prep, we boil the samples with 2-mercaptoethanol in Laemli buffer. However by mistake, some of the...

03 December 2020 7,088 3 View

What does it mean if you have two variables strongly correlated but only one variable changed in the experiment?

Hello I have two variables measured: one is a behavioural variable and one a western blot variable of a protein. They are strongly correlated. However, only the behavioural variable was...

03 December 2020 1,071 3 View

Why the western blot of HIF1alpha of the whole cell lysate shows intense band under normoxia without any induction of hypoxia?

I am working with oral cancer cell lines and trying to find the expressional changes of HIF1alpha under different experimental treatment. I am getting intense band of HIF1alpha in the western blot...

30 November 2020 5,894 3 View

Why does immunofluorescence but not western blot works for fusion protein?

I have a flag-tagged protein and want to be sure it is properly expressing in my cells. When I assess by IF, I see clear nuclear enrichment (expected). But when I do a western, there is only a...

28 November 2020 2,519 3 View

No detection in Immunofluorescence Staining of control while no much difference in the both groups in western blot, why?

We are working on an enzyme (145 KDa). Firstly we did Immunofluorescence staining on the paraffin embedded sections (4 µm thick). No protein was detected in PBS group while high level was...

24 November 2020 8,704 3 View

Band size NFkB of app. 150kDa??

I made a western blot with the p65 NFkB antibody and the phospho p65 NFkB antibody from cell signalling. I get a band for both the phospho and non-phospho antibody at around 150kDa, how can this...

17 November 2020 2,118 3 View

Western double band and si-transfection?

I kept getting double bands when western blotting, one at 25kDa and one at round 30kDa. I thought it could happen because I know that there are two isoforms from this gene. problem cames in here...

17 November 2020 1,434 3 View

How to get rid of the free GFP signal in an Atg8-GFP tagged yeast saccharomyces cerevisiae?

I am working with yeast saccharomyces cerevisiae -BY4741- strain. I tagged the Atg8 with GFP in this background and whenever I am performing western blot I get a free GFP signal for my WT which...

16 November 2020 8,751 3 View

How to detect Stathmin1 with Western Blot?

Hi all, I have problem with Stathmin1 17 kDa protein from mice brain tissue on western blot. Does anybody have experience with Western blott for 17 kDa protein? I am using 12% gel, SDS-PAGE, and...

15 November 2020 6,476 8 View

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