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 do I blot an 8kDa protein?

Hi, I am trying to blot a protein of 8kDa with western blotting. So far I have tried to use 0.45um & 0.2um nitrocellulose membranes for transfer as well as 0.2um PVDF. I have tried to use...

10 October 2019 7,571 5 View

How to distinguish between soluble and insoluble protein fraction?

I am having strange results with my Western blot results. My protein of interest is suppose to increase after treatment. Infact it has decreased compared to the control. Does this mean the...

10 October 2019 4,369 4 View

CRISPR partial gene reduction but protein is still detectable, wth?

Hello everyone, I'm helping an undergrad student who tried to knockout the DUSP2 gene in the PDAC cell line (Mia Paca-2). The RT-qPCR result showed a 60% reduction of gene expression, but the...

10 October 2019 2,252 2 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...

10 October 2019 4,200 4 View

Membrane protein assay?

I'm looking for a technique besides western blot to show the expression level of my membrane protein? A quantifying assay?

10 October 2019 1,362 5 View

Why am I getting weaker signal for samples on the edges of blots-Western blot?

Hi everyone, I have been running into this issue for quite some time. I notice that the intensity of bands that are towards the middle of my blots are always higher compared to the edges. (The...

10 October 2019 3,429 5 View

Shadowy background on my Western Blot ?

I'm doing a western blot with biotinylated antibodies. Everything worked fine until 2 months, a black background appeared on handling with strepta-poly-HRP. My typical protocol: PVDF membrane,...

10 October 2019 2,431 4 View

What can I do when ELISA is not available due to matrix effect?

I'm trying to do ELISA for some molecules in human ascites. Before doing main experiment, I did "spike and recovery test" to examine if there is any matrix effect in my samples. The recovery...

10 October 2019 6,974 3 View

What could cause these "frowning" bands seen in electrophoresis (SDS-PAGE)?

As part of my western blotting procedure, I ran my SDS-PAGE electrophoresis. Usually, I load in 2 lanes, 1 ladder (spectra broad range protein ladder) and my sample (sample lysate + Laemmli...

10 October 2019 2,128 16 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 1,343 3 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?

07 October 2019 1,099 4 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....

04 October 2019 6,404 3 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...

02 October 2019 8,878 4 View

How can I detect cleaved-caspase 3 by western blotting?

Hi everyone, I'm not able to detect cleaved caspase-3 by western blotting using the ab13847 antibody. My wb input are lysed primary cortical neurons treated with staurosporine 1 uM for 4 and 6...

23 September 2019 3,482 3 View

The best voltage and current for LC3 western blotting?

Our power supply for western blotting transfer process needs three parameters of watt, voltage and current. In LC3 western blotting in different methods just the voltage or just the current are...

09 September 2019 9,895 1 View

WB in rat brown adipose tissue?

How to make for calculate quantity of reagents and antibody necessary for realize western blotting in 28 samples of brown adipose tissue?

09 September 2019 5,036 3 View

What is the band, detected by pTrkB-Y515 antibodies on 92kDa?

Hello, I study the change in TrkB phosphorylation under the short-time (no more 1 hour) influence of certain factors in HT-22 hippocampal using Western blot analysis. Antibodies ThermoFisher...

09 September 2019 8,625 1 View

Cell viability using MTT assay in the precence of amyloid beta 1042?

Hi Dear all I used aggregated amyloid beta 1-42 (10 micromolar) to induce Alzheimer's condition in PC12 cell line, but after 24 h incubation, MTT assay was done, I obtained 85% cell viability, I...

09 September 2019 9,606 7 View

Could someone recommend good antibodies for detecting M1, M3 AchR and SERCA1/2/3 in WB and IHC?

I'm working in a project related to Ca2+ signalling and I've to detect protein expression of M1/M3 Acetylcholine Receptors and SERCA1/2/3. Could someone recommend good antibodies for detecting...

09 September 2019 4,202 2 View

What is wrong with my total protein gels??

I am new to western blotting and am practicing the procedure up till the transfer step (I'm good after that step). After the electrophoresis step I take a look at the total protein on the gel, and...

09 September 2019 1,238 3 View

What is this ~35 kD contaminant band in my exosome Western blots?

I have been trying to run Western blots for ALIX and CD63 using SBI's antibodies, but I get big, bright bands at ~35 kD for all of my samples. The samples were isolated using SBI's ExoQuick ULTRA...

09 September 2019 6,461 3 View

How can I normalize Western blots if housekeeping proteins change in time?

Hello, I am evaluating the expression pattern of some proteins during the process of neuronal differentiation (1 day in vitro to 32 days in vitro) in primary neuron culture using western blot. I...

09 September 2019 4,633 3 View

Tissue protein quantitation for western blot by Coomassie brilliant blue G 250?

Anyone can share the protocol to measure mice tissue (spleen) protein concentration for western blot by using Coomassie brilliant blue G 250 and how to calculate from results the amount of sample...

09 September 2019 5,085 4 View

Question about western blot conditions with antibody-COL1 (ab6308) ?

Dear Sir/madam I have bought the anti-COL1 (ab6308). it is an antibody that is only reactive with the native form of the protein and works under non-reducing, non-denaturing conditions. I find it...

09 September 2019 786 3 View

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