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

1,952 views 231 posts

Questions related to Immunoblotting

I can't detect any protein in my Western blot. Does anyone know how to fix this?

I'm doing a Western blot and haven't seen any protein for two months. After transferring, the marker is visible, but when I detect the protein once, wash it, and try to detect another protein the...

11 June 2024 6,595 7 View

Do you have beta actin primer set seq?

Hello, Do you have beta actin primer sequences for qPCR? I will study with 3T3 (mouse fibroblast), Raji (Human B cell), U937 (Human monocyte), HEK293 (Human embryonic kidney). May you suggest...

01 December 2022 9,242 0 View

Can anyone suggest good primer for B-actin,Npy, AgRp for RT-PCR?

Hello research enthsiasts! I want some suggestion on good primers for RT-PCR for a)beta actin b)Neuropeptide Y c)Agouti related Peptide Please also mention the journal for reference? High impact...

21 October 2022 666 2 View

Are there Hif1a antibodies that work reliably for immunofluorescence on frozen sections?

A few years back I tried several available Hif1a antibodies on frozen sections of heart and lung. None of them worked very well. Most worked well for immunoblots. Have there been any advances in...

13 October 2020 7,886 2 View

What is wrong with this western blot?

Dear fellow researchers: I've recently started performing WB, mostly with molecular weight markers to test the equipment and optimize I/V conditions. This time I also ran protein lysates from two...

13 August 2020 4,144 11 View

On my western blotting/ECL, using anti-alpha tubulin, aside the 55kDa alpha tubulin band, extra band appears at 35 kDa, wondering wht this band is?

When using abcam anti-alpha-tubilin on immunoblotting developed on ECL to probe tubulin on mice splenocytes, I see a clear band at 55kDa but there appears consistently another band at about 35kDa,...

27 July 2020 2,301 4 View

Can anyone explain why do we get diffused bands in western blot? and any trouble shooting techniques?

Hello, Everyone. I have some questions related to western blot as I am new to western blot. I tried to detect the LC3B, however I got diffused bands as I have attached in the file (Please find the...

13 November 2019 8,404 13 View

How much should we add primary antibody into 20 mL of 1% Skim milk for Western Blot?

Hello, Everyone! I currently start doing western blot experiment, but I am not really sure how much should I add primary antibody into 20mL of 1% Skim milk. Here is what I am doing: * I make 1%...

11 November 2019 5,372 9 View

How to interpret this LC3 immunoblotting result for the autophagic function?

I performed an immunoblotting with LC3 antibody (please see the image) upon siRNA-mediated knockdown of two different proteins, A and/or B, with or without Bafilomycin A1 treatment. I would like...

21 October 2019 8,664 4 View

Does an increase in reactive oxygen species (ROS) indicate apoptosis?

Our combination treatments cause a reduction in cell number and a 2 fold increase in ROS but our other experiments do not suggest apoptosis: no cleavage of Parp, or increase in Bad expression by...

10 October 2019 8,824 8 View

How to interpret this LC3 immunoblotting result for the autophagic function?

I performed an immunoblotting with LC3 antibody (please see the image) upon siRNA-mediated knockdown of two different proteins, A and/or B, with or without Bafilomycin A1 treatment. I would like...

10 October 2019 8,211 5 View

What is an appropriate marker for matured adipocytes?

Hello, I recently isolated protein from adipose-derived stromal vascular fraction. I hope to use this for immunoblotting. Is there a marker to sort of confirm that this fraction isn't...

08 August 2019 3,837 1 View

Do you know of any affordable technique or service to identify cross reacting proteins with a specific antibody?

I am facing with a perplexing situation in which there are two proteins both identifying correct protein sizes in immunoblot by polyclonal antibody (monoclonal doesn't exist in market) but it is...

07 July 2019 6,052 4 View

Recombinant protein for antibody neutralization?

I need to understand if an antibody I'm using is specific or not. The antibody is not validated. In immunoblotting, the antibody detects a prominent band at the right molecular weight and two...

30 April 2019 4,922 4 View

Does the technique of measurement impact the final Kd of enzyme towards its substrate?

I am trying to understand if, two different ways of performing enzyme binding kinetics may impact the final Kd values and if yes to what extent for slow and fast reactions. For more context...

18 March 2019 4,030 3 View

Does the technique of measurement impact the final Kd of enzyme towards its substrate?

I am trying to understand if, two different ways of performing enzyme binding kinetics may impact the final Kd values and if yes to what extent for slow and fast reactions. For more context...

03 March 2019 9,130 5 View

Need to use Protein A deleted strain for whole cell immunoblots for S. aureus or not?

I found a lot of paper using whole cell dot immunoblots to detect surface protein. Antibody may be specific IgG or serum, and working with wildtype of strain. But protein A will bind to any IgG,...

11 February 2019 6,775 3 View

Deglycosylation test: I can't understand why the bands from my deglycosylated proteins are not neat?

I carried out a deglycosylation test to see if there will be difference in the electrophoretic mobility of C57BL/6 infected-ME7 and SShpPrP Tg ( transgenic mice expressing sheep prion protein)...

02 February 2019 5,939 2 View

Western blot on immunoprecipitation eluate detects related protein?

Hello, So, first I perform immunoprecipitation. The eluate is then subjected to immunoblot analysis using an antibody against a related protein (equal size) and gives a band. How sure am I the...

14 November 2018 5,500 7 View

Western blot on immunoprecipitation eluate detects related protein?

Hello, So, first I perform immunoprecipitation. The eluate is then subjected to immunoblot analysis using an antibody against a related protein (equal size) and gives a band. How sure am I the...

11 November 2018 4,611 8 View

Hello everyone. Please tell me that what amount of estrogen and estradiol should be used for MTT and immunoblotting assay?

Estrogen and estradiol, MTT and immunoblotting assay

10 October 2018 2,382 0 View

How to raise primary antibody against my protein of interest?

I want to raise primary antibody against my protein of interest in my lab. I need help to make it. I have full length mRNA sequence of my gene. Can you provide me any protocol to raise primary...

09 September 2018 9,039 6 View

CO-IP with FLAG tag and HA tag?

I have been trying to do a co-IP using FLAG tagged and HA tagged proteins. Did another western after transfecting each 1XHA -A protein(55kD) and 3X FLAG-B-protein(69 Kd) in separately and they...

27 June 2018 2,095 8 View

Why monoclonal and polyclonal antibodies, targetting the same protein, reveal immunoblotting bands with different molecular weights?

Hello, I am using two different antibodies specific for the same membrane protein: one of them is monoclonal and the other polyclonal. However, when I reveal the PDVF membrane the results are...

06 June 2018 1,335 4 View

Badges
Science method