Questions tagged [western-blot]
Questions pertaining to western blots, which are used for detecting specific proteins (sometimes called protein immunoblots).
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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 ...
8
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1answer
491 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 ...
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2answers
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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 ...
5
votes
2answers
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 ...
5
votes
1answer
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 ...
5
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1answer
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 ...
5
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1answer
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-...
5
votes
1answer
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 ...
5
votes
2answers
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 ...
4
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2answers
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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 ...
4
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1answer
203 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 ...
4
votes
1answer
459 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 ...
4
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1answer
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 ...
4
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3answers
437 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 ...
4
votes
1answer
437 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 ...
4
votes
1answer
54 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 ...
4
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0answers
296 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 ...
4
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0answers
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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 ...
3
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3answers
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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 ...
3
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1answer
758 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....
3
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1answer
245 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-...
3
votes
2answers
23k 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:
...
2
votes
2answers
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 ...
2
votes
1answer
921 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 ...
2
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1answer
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.
2
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1answer
137 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-...
2
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1answer
190 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 ...
2
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1answer
43 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 ...
2
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0answers
163 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. ...
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vote
1answer
2k 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, ...
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vote
1answer
31 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 ...
1
vote
1answer
17 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 ...
1
vote
1answer
47 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 ...
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vote
1answer
107 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 ...
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vote
1answer
191 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 ...
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vote
0answers
16 views
How to combine data points from different western blots into one graph?
I am doing Western blot data analysis and I have data from two different western blots. In my two Western blots, I have labelled for the same proteins. I am comparing knockout (KO) samples against ...
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vote
0answers
40 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 ...
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0answers
89 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?
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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 ...
0
votes
1answer
37 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 ...
0
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1answer
35 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 ...
0
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1answer
35 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?
0
votes
2answers
70 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 ...
0
votes
1answer
204 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.
0
votes
1answer
63 views
Molecular weight of my 2-D gel
I am a little confuse when I try to figure out the molecular weight of the marker on my gel. I used NuPage Novex 4-12% bis-tris gel and Mark12™ Unstained Standard as a marker. Could please someone ...
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0answers
18 views
Question about total protein normalisation in western blot image analysis
I am analysing western blot data and I am using total protein normalisation using Ponceau S. My supervisor and I have agreed to use total protein normalisation for my western blots instead of using ...
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0answers
8 views
In Western blot, is Ponceau S a valid loading control if band intensities vary between lanes?
I have done a Western blot and I am using Ponceau as a loading control to normalise my measurements against a protein of interest. For my Western blot I have a number of wild-type and knockout samples ...
0
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0answers
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Western blotting: what common tweaks do you make to the protocol and why would you make membrane cuts?
I'm building an app which helps researchers plan out Western blots, record protocol tweaks and receive warnings in the app if:
you're trying to multiplex antibodies of similar molecular weight on the ...
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0answers
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In a Western blot, when analysing phosphoprotein bands can you normalise against total protein or do you need to use a loading control?
I am doing Western blot data analysis and I have probed my membrane with phospho-specific antibodies against a phosphoprotein (it is a protein kinase) of interest. I have also probed my membrane with ...
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0answers
17 views
Recommendations on books about statistical analysis for Western blot data?
I have run a couple of Western blots and I am planning on doing more in the future. My goal is to combine the results from the different Western blots and perform a statistical analysis in order to ...
