Questions tagged [western-blot]

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

11 questions with no upvoted or accepted answers
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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 ...
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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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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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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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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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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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20 views

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 ...
-1
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17 views

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 ...
-1
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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 ...
-1
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1answer
45 views

In western blots, do loading controls have to be proteins that are in equal amounts in each lane?

I have recently done a Western Blot and I am doing data analysis on my blots. I am studying the protein GSK3, which is a phosphoprotein. I have labelled my membrane against active GSK3 and inactive ...