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

How to get Proteinase K resistant bands of PrPSc from ScN2a lysate by western blot?

I have already tried different lysis buffers and many protocols also. But, still I am not getting consistent result.

02 February 2020 8,544 3 View

Why there are multiple bands in Western blot after protein expression in insect cells?

Hi, I recently used an old P0 Baculovirus (stored at 4C for 8-9 months) to produce a viral glycoprotein (37kd). My previous expression for this protein using SF9 cells was always very low so I...

02 February 2020 3,806 6 View

How important is it to include the densitometric analysis of Western Blot (WB) data in the article?

I found in several articles where the authors use the quantitative analysis (densitometric) of the WB bands, where in most cases the significant data is not found. I think that obtaining...

02 February 2020 6,835 5 View

Best approach for protein extraction from C. elegans?

Hello. I need to detect a protein that is present only in larvae (and compare them to adults). The problem is that I want to load equal amounts of protein. And I want to make Bradford assay. I...

02 February 2020 4,203 3 View

Loading control for Western Blot on NRK49F cells +TGFb1 ?

Hi all, I encountered an issue with my western blot : the loading controls I tried were all down regulated after 24h TGFb1 stimulation in NRK49F cells (rat kidney fibroblasts) so I can't use the...

02 February 2020 3,720 2 View

What the difference to do Western Blot using Protein Simple system?

I just moved to my new laboratory, and I want to do Western Blots. I am used to the Bio-Rad system. Since I have to set up the things here. I saw that is possible to `Western Blot` using protein...

02 February 2020 7,360 1 View

Protein concentration and number of sample for western blots?

Hello All! I want to test protein expression in 5 different type of muscles (with different morphology) . I have homogenised a varied number of samples per each muscles to increase protein...

02 February 2020 3,077 13 View

How to perform a western blot of a sample containing 50% Metanol?

Hi there, I am using cold methanol fixation (1 volume cold methanol to 1 volume cell culture medium) and then lyse my cell. Since method damages the cell membrane am wonder whether my protein...

02 February 2020 4,531 4 View

Weird black spots on western blot... potential causes?

I ran BeWo lysate samples on a polyacrylamide gel in SDS under reducing conditions. Gel was transferred to nitrocellulose membrane and blocked with 5% nonfat milk/PBST. Primary and secondary...

01 January 2020 8,701 5 View

Is it high molecular weight or aggregated proteins?

I am still confused about the upper bands in most of my western blots, is it aggregated protein or high molecular weight?

01 January 2020 671 4 View

Aspecific bands in Western Blot, what are they and how to get rid of them?

Dear all, I am performing Western Blots on primary HUVEC cell lysates, derived from cell cultures with fibronectin coated plates and Lonza EBM-Plus medium. I used RIPA buffer for lysis. I...

01 January 2020 4,708 4 View

How to optimize Western Blot of exosomal markers?

I am isolating exosomes from human plasma using the IZON SEC column. I want to detect exsomal markers Flotilin-1, CD9, HSC70 and TSG101 in my samples. I am using SDS -Page and PVDF membrane, all...

01 January 2020 2,727 5 View

Cutting Western blots with proteins with similar molecular weights?

Hello everyone, Currently, I'm trying to detect five different proteins on a single western blot gel. To do this, I load 5 of the lanes with my sample lysate and I cut it vertically along each of...

01 January 2020 3,606 2 View

What can I use to wash my primary neurons before I add RIPA?

Hi! I have a technical question concerning protein extraction from neuronal primary cultures. When I grow glial cells in the wells (coated with PLL), after I aspirate the supernatant I usually...

01 January 2020 1,965 3 View

Has anyone detected amino acids using Western blots?

Hi all, I am trying to find a method to measure amino acids such as methionine and homocystiene in a fluid. We have used dot blots in the past but I am wondering if anyone has successfully used...

01 January 2020 1,979 3 View

SDS-PAGE & tRNA associated proteins?

Hello, I am currently reading this article by Lin et al, 2020 (https://science.sciencemag.org/content/367/6473/100) about this TTC5 enzyme and its new-found role in tubulin mRNA regulation. I...

01 January 2020 7,083 2 View

Why I am having a dark area in the upper part of my blot?

Hello, Recently, I'm having this strange dark area in the upper part of my western blot. And it happened many times in the same area exactly!! I think it is a running problem, but I can't...

01 January 2020 9,595 3 View

How to compare data from different Western Blots?

Hello everyone, I want to compare data from different western blots. Which method do you think is the most appropriate? (normalization by controls/ total proteins/ housekeeping/scanning parameters)

01 January 2020 8,036 5 View

What is the max. conc. of compound 991, which can be used?

I am using ex229 (compound 991), (10 µM), as +ve control for AMPK activation (phosphorylation) in J774 cells (macrophages), but it is not working (Western Blot). It is showing +ve effect on p-ACC...

01 January 2020 7,538 1 View

Problems with using PVDF membrane in a Western Blot?

To conduct my western blot, I used a PVDF membrane which I activated by wetting the membrane in methanol for 1 minutes ( change from opaque to translucent), placing the membrane in Milli Q water...

01 January 2020 10,106 5 View

Can I examine chemokines using a western blot from total endothelial cell lysate?

After qPCR analysis of HUVEC lysate from my experiment, I found upregulation of several chemokine mRNA that I would like to validate via western blot. Due to the nature of my experiment, it would...

16 December 2019 5,438 3 View

What happened to my cells after nocodazole treatment?

After nocodazole treatment for cell cycle synchronization, most of my cells became round, but didn't float. What happened to them? Can I continue to lyse them for coIP and western blot?

12 December 2019 4,859 9 View

Do I need to validate all protein results from my tandem mass spec experiment?

Hi, I am looking to run a tandem mass spec experiment for proteomics. Obviously I will need to validate the results of this using another method such as Western Blotting/Elisa. The question is,...

12 December 2019 4,238 2 View

I cannot detect the western blot band of VDAC1, which step maybe the problem?

I cannot detect the western blot band of VDAC1, a Mito marker, in the whole naive T cell , but postive sample (VDAC1 protein) band is very clear. I use RIPA to lyse naive T cell (about 6 M...

12 December 2019 7,661 3 View

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