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

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8
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1answer
279 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 ...
5
votes
2answers
498 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 ...
5
votes
1answer
490 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
556 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
votes
2answers
116 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 ...
4
votes
2answers
194 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 ...
4
votes
1answer
106 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 ...
4
votes
1answer
127 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 ...
3
votes
1answer
149 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 ...
3
votes
3answers
105 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 ...
3
votes
1answer
281 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 ...
3
votes
1answer
103 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 ...
3
votes
1answer
39 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 ...
3
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0answers
27 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 ...
2
votes
1answer
48 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 ...
2
votes
1answer
408 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 ...
1
vote
1answer
113 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 ...
1
vote
1answer
59 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 ...
1
vote
3answers
61 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 ...
1
vote
1answer
68 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 ...
1
vote
1answer
74 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 ...
1
vote
0answers
37 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. ...
0
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0answers
55 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 ...