Questions tagged [western-blot]

Questions pertaining to western blots, which are used for detecting specific proteins (sometimes called protein immunoblots).

Filter by
Sorted by
Tagged with
10 votes
1 answer
2k views

Mass spectrometry versus western blotting for validation

I have mass spectrometry data (LC-MS/MS) from rat cortices under either drug or control treatments. The results were performed in triplicate (three pairs of rats, drug or control per pair). In ...
user avatar
  • 345
8 votes
1 answer
514 views

Optimal pH of protein buffer? Basic principles to adjust buffers according method and analysis

Protein buffers such as PBST, which is used in western blotting, are normally adjusted to pH 7.4. When I try to find why, I find some information about optimal pKa for protein stability. Im not sure I ...
user avatar
7 votes
2 answers
2k views

Can this image be the result of a Western Blot?

I am currently researching image manipulation in the life sciences and stumbled upon a Western Blot looking very similar to the image below. For creating the image I have cut out the blot using the ...
user avatar
5 votes
2 answers
3k views

Why proteins are not visible on my membrane after ponceau staining?

I have a problem in western blot that I can't resolve by myself. When I am use to add 100 microgram of proteins for each sample but after running and transfer its impossible to me to see bands of ...
user avatar
  • 123
5 votes
1 answer
16k views

How to prepare sample for SDS PAGE?

I usually take my protein sample 0.8ml and sample buffer(2X) 0.2ml for my sample preparation in SDS PAGE. Am I using correct proportion? my protein sample concentration is 4.1mg/ml. What is the ...
user avatar
  • 229
5 votes
1 answer
2k views

Are there any disadvantages to HRP conjugated PRIMARY antibodies

I need to detect a FLAG tagged protein on a western blot and have to order an antibody. I'm not expecting to see a massive amount of my protein. To keep analysis time to a minimum I am considering a ...
user avatar
  • 1,405
5 votes
1 answer
1k views

Western blotting with multiple antibodies

Normally I wash/detect with one primary/secondary-HRP antibody pair, strip, then wash/detect with the other primary/secondary-HRP pair which works well. However, I recently started working with a HRP-...
user avatar
  • 3,264
5 votes
1 answer
1k views

How to do western analysis to lung tissue?

I am working with lungs from mice, and I want to do a western blot analysis to my samples. I am having trouble with this because my images turn out very "messy", with a high background. Perhaps I ...
user avatar
  • 191
5 votes
2 answers
1k views

High Current (Speed) Transfer Buffer Recipe

Does anyone know an effective buffer mix to use for high current Western transfers? We are successfully using the vendor's premixed buffer to transfer a wide range of protein sizes to PVDF membranes ...
user avatar
  • 4,089
4 votes
2 answers
188 views

Is there a strong reason to be sceptical about the "cured HIV patient" being reported by mainstream media?

There's a story going round the news about a baby that was, apparently, cured of HIV using a cocktail of drugs at an early age. The story piqued my interest, but details seem scarce. One of the main ...
user avatar
4 votes
1 answer
259 views

What are the units of the band intensities in a western blot image?

I have done a Western blot and I am measuring the band intensities in Image J. Using the rectangle tool, I have outlined the band and then calculated the area and the mean intensity of the band. I ...
user avatar
  • 1,561
4 votes
1 answer
516 views

How to remove bad lanes in ImageJ Westernblot analysis

I use ImageJ to do an analysis of a Westernblot Image. If everything goes as wanted the workflow is fine. But if I do something wrong creating a lane there is no undo for a lane and also no way to ...
user avatar
  • 91
4 votes
1 answer
2k views

Western blotting questions?

everyone. I've just been introduced to the procedure of Western blotting from my reading, though I'm not entirely sure about some points. I'd appreciate it if someone could help me with this. What ...
user avatar
  • 169
4 votes
3 answers
449 views

Western Blot: Fischer ECL substrate 2:1 instead of 1:1?

i recently developed a b-actin-ab-incubated PVDF-WB-Membrane with the Thermo Scientific Pierce ECL Kit. According to the manufacterers orders i mixed Reagent 1 and Reagent 2 1:1. But unfortunately i ...
user avatar
4 votes
1 answer
445 views

What equation to compare protein isoforms in a Western Blot?

The protein isoforms I am interested in comparing appear as distinct bands on the gel I have already run. I have an Excel sheet with optical density measurements I obtained using ImageJ; it looks ...
user avatar
  • 1,020
4 votes
1 answer
55 views

Prevalent large (>=90kDa) maintenance protein/loading control

I was wondering if anyone had recommendations for good, large (hopefully 100kDa+), control proteins that would be present in most mammalian cells. I'm working mostly with tissue samples from humans ...
user avatar
  • 4,089
4 votes
0 answers
311 views

Relative densitometry from SDS PAGE

I'd like to perform densitometry on a Coomassie stained SDS PAGE gel to compare a recombinant protein's expression levels under two conditions. I'm using BioRad's Image Lab software. My questions are ...
user avatar
4 votes
0 answers
87 views

Procedure for doing western blot [closed]

I am writing a step by step guide for doing a western blot for a class. It is intended for any one with basic Biology lab skills. I am hoping people will review my draft and give feedback on how to ...
user avatar
3 votes
3 answers
1k views

LCMS/MS versus Western Blot

I have a general question regarding which method would you recommend me to use if I would like to investigate the difference in the level of several proteins in tissue samples and compare different ...
user avatar
  • 31
3 votes
1 answer
861 views

Western blot alternatives

Given an in vivo sample from an experimental infection, I would like to see if a bacterial protein is present. I've been thinking about Western Blot using sera from animals infected with the bacteria....
user avatar
  • 961
3 votes
1 answer
260 views

Western blot transfer issues

I am having some issues with transfer efficiency to nitrocellulose membranes. To get all the technical info out of the way: Transfer method: Wet, tank transfer overnight at 30V with normal tris-...
user avatar
3 votes
2 answers
26k views

What is the role of glycine in the running buffer for SDS-polyacrylamide gel electrophoresis

We used pH6.8 in stacking and pH8.8 in resolving gel. In the class, the professor explained that the glycine change is like: ...
user avatar
  • 195
2 votes
2 answers
9k views

Why is beta actin commonly used as a control in Western blots?

It is about the western blot question. The paper:http://www.sciencedirect.com/science/article/pii/S1931312813001145. In the western blot experiment in this paper, they use beta actin as control. And ...
user avatar
  • 195
2 votes
1 answer
1k views

Can I use grayscale images when working with ImageJ?

I am using ImageJ to analyze Western Blots. I have scanned films in as grayscale images because this is how we did it in my old lab. People in my current lab are not satisfied with that explanation ...
user avatar
  • 1,020
2 votes
1 answer
87 views

Can Western Blots be used to quantify the activity of a protein?

I am new to Western Blot analysis and I have recently done my first two. I am studying a phosphoprotein (a protein kinase) that can be both activated and inactivated via phosphorylation at a specific ...
user avatar
  • 1,561
2 votes
1 answer
21 views

What for a Kinase Assay? [closed]

I am wondering why to use a kinase assay, since we can extract the proteins from cells and then do a Western with the specific antibodies we want to use.
user avatar
2 votes
1 answer
141 views

Why does my anti-ubiquitin antibody visualization not work on my PAGE gel?

I am using 2D gel electrophoresis to visualize polyubiquitinated proteins. However, while I can see actin and heat shock protein using when appropriate antibodies, I cannot visualize them using anti-...
user avatar
  • 227
2 votes
1 answer
201 views

can a bacterial protease inhibitor cocktail be used for Western Blotting involving HUVEC cells and HT-29 adenocarcinoma cells?

Dear fellow biochemists, I need some advice on Western Blotting, more specifically the use of certain protease inhibitors with the RIPA cell lysis buffer and protease inhibitor cocktail. A Millipore ...
user avatar
2 votes
0 answers
21 views

In phospho/pan analysis in Western blots, what is best way to normalise to an internal loading control?

I am analysing the expression of a protein kinase X that is a phosphoprotein through Western blots. I have labelled the membrane for both the phosphorylated form of the protein and also for the total ...
user avatar
  • 1,561
2 votes
0 answers
184 views

Why HIS tag is not detected in WB or dot blot?

I would like to know why the recombinant protein with his tag at c terminus is not being detected neither in western blot or dot blot. I'm trying to validate protein expression of four proteins. ...
user avatar
  • 961
1 vote
1 answer
3k views

PBST vs. TBST buffer in western blotting

What is the advantages and disadvantages of using either PBST or TBST in western blotting, or while working with proteins in general? Are there other buffers which are also used for western blotting, ...
user avatar
1 vote
1 answer
39 views

Western blot trouble shooting - low voltage and yellow sponges during/after transfer

I have a western blot troubleshooting question that I haven't been able to find the answer to in manufacturer troubleshooting guides. As a bit of background, I was transferring 2 western blots ...
user avatar
1 vote
1 answer
36 views

Need help interpreting Western blot data

So the paper I am reading (here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094582/) uses densitometric units to quantify the western blots. The authors mentioned that the unphosphorylated proteins ...
user avatar
1 vote
1 answer
123 views

Can you use images taken at different exposure times in western blot image analysis?

I am doing Western blot data analysis where I have images from a number of experiments (where the samples in the experiments are biological/technical replicates). For each experiment, I labelled the ...
user avatar
  • 1,561
1 vote
1 answer
43 views

What is the best way from remove background signal from a band when doing Western blot image analysis?

I am doing image analysis of a Western Blot in Image J. I have calculated the total intensity of my protein bands of interest through outlining each band using the rectangle tool in ImageJ, and ...
user avatar
  • 1,561
1 vote
1 answer
58 views

Is it necessary to calculate lane normalisation factor when doing western blot data analysis?

I have recently done my first western blot and I am doing data analysis to quantify my blot. I have labelled my membrane against inactive GSK3 and active GSK3 which are phosphoproteins so I am using ...
user avatar
  • 1,561
1 vote
1 answer
110 views

Use of western blotting

I ran across this paper recently. http://www.ncbi.nlm.nih.gov/pubmed/21151122 I am confused on their use of Western blotting. Their unmodified protein (no PTM group) has a band and their modified ...
user avatar
1 vote
1 answer
197 views

Enzymatic Protein Deglycosylation of TCL

I've done O- and N- linked enzymatic deglycosylation of purified and lyophilized proteins. Following some modifications to the manufacture's protocol, I had great results. I now want a student to ...
user avatar
  • 4,089
1 vote
0 answers
45 views

Why do you need to calculate the lane normalisation factor when doing western blot data analysis?

I am learning about western blot data quantification from some online resources. I have read about methods to normalise the data. I have seen in a number of resources, such as this handbook and this ...
user avatar
  • 1,561
1 vote
0 answers
43 views

Problem with Caspr antibody in western blotting

I have been working on optimizing western blotting for Caspr (contactin associated protein), but I am having difficulties and would like suggestions. I ran my proteins on a 40% acrylamide gel, I ...
user avatar
1 vote
0 answers
92 views

Using only one antibody to detect COX-2 enzyme

If I wanted to detect COX-2 from a western blot test, would only a primary antibody work? I'm on a budget so does anyone know of relatively cheap COX-2 antibodies?
user avatar
1 vote
0 answers
64 views

Western blotting of lung samples [duplicate]

I have been working on lung samples to perform western blotting. I have two related questions about this: I get very nonspecific results: at times nothing at all, at times it glows on membrane but ...
user avatar
0 votes
1 answer
97 views

What is meant by 4 –12% or 8% SDS-PAGE?

I am reading a journal paper and I am looking at the materials and methods section. Regarding the Western blot method in the paper, I have come across the following statement: Proteins were separated ...
user avatar
  • 1,561
0 votes
1 answer
31 views

In enhanced chemiluminescence in western blots, will the horseradish peroxidase eventually get used up?

I am learning about enhanced chemiluminescence in Western blots. I have read online that in enhanced chemiluminescence that horseradish peroxidase (HRP) catalyses the oxidation of luminol to 3-...
user avatar
  • 1,561
0 votes
1 answer
32 views

Why is it good practice to prepare western blot samples in pairs when you have two experimental groups?

I have done some Western blots where I have analysed the expression levels of a certain protein in brain homogenates from wild-type and knockout mice. When preparing brain homogenates, I was advised ...
user avatar
  • 1,561
0 votes
1 answer
64 views

When analysing phosphoproteins via Western blot, why is total protein level of the target protein recommended as an internal loading control?

I am analysing the expression of a protein kinase via Western blots, and it is a phosphoprotein. I have labelled my membrane with antibodies against the phosphorylated form of the protein (using ...
user avatar
  • 1,561
0 votes
1 answer
44 views

What is background signal in a Western blot?

I have done a western blot and I want to remove the background signal when doing densitometry analysis of my protein bands of interest. I have read some articles online such as this one regarding high ...
user avatar
  • 1,561
0 votes
1 answer
40 views

AMPK, PAN-AMPK, western blot

What does it mean by adding PAN before the AMPK. I am trying to do a western blot assay on AMPK and I am confused by PAN-AMPK, AMPK. does it mean pancreases? Is pan-ampk the total ampk?
user avatar
0 votes
2 answers
187 views

Preparing sample for SDS PAGE

I have more than 10 cell lysate samples (70 µL each) whose concentration varies from 1.9 mg/mL to 4.8 mg/mL. I have 5X and 2X SDS sample buffers. I would like to prepare SDS PAGE samples in such a way ...
user avatar
0 votes
1 answer
271 views

What is the role of triton in buffer for western blot assay?

What is the role of triton in buffer for western blot assay? I understand it has to do with preventing non-specific binding of the antibody.
user avatar
  • 1