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

Which proteins are continuously expressed (at stable levels) in mammalian monocytes and thus, suitable controls for Protein expression studies?

Hello! Currently I am testing whether an externally added agent affects the expression of only one particular protein or whether it generally inhibits protein synthesis due to its toxicity. For...

15 September 2020 6,513 3 View

Why no expression is seen in western blot for a C terminally tagged GPI anchored protein?

Hi,

14 September 2020 9,806 2 View

Beta-metacaptoethanol in Sample Buffer (Western Blot)?

I'm working on Western Blot and was guided to use the Sample Buffer Solution (2ME-) (x4) (Fujifilm Wako). However, Beta-metacaptoethanol (BME) is said to be necessary to cut the disulfide bond in...

14 September 2020 5,667 3 View

Does DTT affect cell lysis?

Hi, I am currently working with S. cerevisiae. general rule of thumbs for yeast cell lysis is to use 0.2M NaOH incubation followed by lysis using high urea buffer (containing 100mM DTT) at 70oC...

06 September 2020 7,176 3 View

How can adenovirus be inactivated in cell lysates prior to western blotting?

I need to collect cell lysates from cells treated with an adenovirus (human serotype 5) and typically use a denaturing lysis buffer such as RIPA. However, I need to inactivate any remaining...

03 September 2020 3,374 1 View

Resuspension mutant huntington (mHTT) in conditioned medium?

Hello all. In lab we grow cells to secret mHTT in media that we then collect and subject to centrigufation at 100k G for 2 hours to concentrate the protein and other secreted components of the...

31 August 2020 2,004 1 View

Which are the best loading controls for Western Blot excluding the cytoskeleton ones?

I need to use a loading control for Western Blot that neither includes a cytoskeleton protein or a protein that interacts directly with the cytoskeleton. My issue is that I incubate cells with...

28 August 2020 2,862 11 View

What is the best protocol of western blot for protein detection and using PVDF membrane?

i am planing to look at alpha glycosides but I I have PVDF membrane and I will use semi-dry method but how I should do western blot and how prepare blocking buffer after running the membrane in...

28 August 2020 6,373 3 View

Delta Furin SARS-COV-2 mutant Western Blot Analysis showed double band around 250 kDa. Processed and unprocessed version? Need evidence, please help?

I analysed SARS-CoV-2 Spike Protein delta Furin mutant in my bachelors thesis and need evidence (paper, research or something like that) to proof my suggestion. I think the double band at around...

27 August 2020 1,188 4 View

Can my RNA interference have any influence in my endougenos control gene?

Good night all. I'm using RNAi to knockdown a transcription factor from a primary cell and I need to validate by westernblot. With Ponceau marking i can see that i'm putting the same amount of...

26 August 2020 406 3 View

Multi band on western blot for glucocorticoid receptor. Which band(s) should I select for quantitative study?

Hello I can see different bands for phosphorylated glucocorticoid receptor protein on the blot membrane. I get confused which band(s) should I select? Can I select the whole areas where the bands...

18 August 2020 6,928 8 View

LC3 II form remains unchanged but p62 protein is increasing, why?

Hello, I am treating my cells with a compound to study whether the autophagy can be affected or not. Using Western blot, I found that LC3 II form remains unchanged but p62 protein is increasing ....

17 August 2020 9,057 3 View

Does anybody have experience with the detection of a FLAG epitope on a Western blot with the Sigma Anti-FLAG antibody M2 after stripping the blot?

I stripped several western blots with 62.5mM Tris-HCl (pH 6.8), containing ß-mercaptoethanol and 2% SDS for 1h at 50°C. After this the FLAG epitope bound to my protein was not detectable anymore...

14 August 2020 8,530 3 View

What do you suggest to investigate cell signaling with the small number of cells?

I have a plan to investigate cell signaling on NKG2D-expressing CD4. For this matter, I plan to isolate primary cells from patients and use western blot technique to measure the expression level...

09 August 2020 1,065 5 View

Western blot in brown adipose tissue?

Good morning, I am a PhD student trying to get my head around brown adipose tissue western blot. So far I have successfully isolated proteins with high molecular weight such as PGC1a but I keep...

02 August 2020 8,934 3 View

Does anyone have a protocol about growing 3D culture in matrigel in order to extract protein for Western Blot assay?

Hello. I'd like to perform a Western Blont with samples obtained from 3D culture in matrigel. I've done experiments with 3D cultures in 96 well plates in order to test drug activity, but now I'm...

28 July 2020 1,592 4 View

How do I interpret these western blot results?

Hi! I got this western blot of the heat shock protein hsp70, and I need to interpret the result. The western is made on 2 individuals A and I and is shocked 5 minutes and 15 minutes, plus there is...

18 July 2020 285 5 View

AMOS PROBLEMS : he model is probably unidentified.Do you have any suggestions ?

Hello AMOS connaisseur. I have a problem with my model. I'm doing a CFA, using the bifactor model to validate a new scale with 7 factors and a general factor. I tried to run the model but got this...

11 July 2020 4,998 3 View

Best GST antibody for Western blot?

Hi, I am performing GST purification, however, I am getting poor signal with my GST antibody at the recommended dilutions -...

12 June 2020 9,905 1 View

MAb detecting two viral proteins?

Hi, What are the chances that the developed MAb can detect two viral proteins simultaneously? The MAbs were raised against purified viral particles. During the characterization studies, some...

29 May 2020 4,196 1 View

Why am I unable to detect Histone PTMs at their correct molecular weight in nuclear extracts via Western Blotting?

I am trying to detect changes in Histone PTMs with or without treatment in nuclear extracts from cell lysates. I use a Hypotonic buffer (20 mM Tris pH 7.4, 10mM NaCl, 1mM MgCl2, 0.5mM EDTA)...

11 March 2020 2,800 6 View

What is the observed molecular weight of Immediate early gene Egr-1 in mouse cortex whole cell lysates?

Hi everyone, I used anti-Egr-1 (rabbit monoclonal, ab133695, Abcam) in western blotting experiment and I see multiple bands ranging from 50kDa to 130kDa, where at 130kDa bands are more intense...

03 March 2020 3,158 1 View

Ways to reduce background noise for Western Blot ?

Hi everyone, Currently, I am running a western blot where I cut my membrane into 5 different pieces and individually incubate them with their respective primary and secondary antibodies. I have...

02 February 2020 6,884 9 View

How many incubations can a western blot membrane undergo before having to do a stripping step?

I'm interested in incubating my membranes with several antibodies and wonder how many incubations a membrane can undergo before having too much background and non-specific binding. Usually I...

02 February 2020 931 4 View

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