8 votes
Accepted

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

The units in your case are arbitrary units. For instance, you may have taken a photo of the bands with a camera that saved a gray-scale file with an 8-bit depth. That means that the brightness values ...
S Pr's user avatar
  • 6,202
4 votes
Accepted

PBST vs. TBST buffer in western blotting

I used to work for a company well-known for its modification state-specific antibodies, including phospho-specific ones, and they actually performed extensive in-house testing of PBST vs. TBST in ...
MattDMo's user avatar
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3 votes

Does beta-actin have to be consistent in Western Blots?

The long and the short of it is that your actin bands do not need to be consistent so long as you can normalize and have met the quite stringent conditions for normalization to be effective. For ...
bob1's user avatar
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3 votes

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

Thank you for the comments. When I got back into the lab I double-checked the chemical shelf and it turns out I had put NaCl into the buffer instead of glycine! Currently soaking the sponges over the ...
Ryan Ellison's user avatar
3 votes
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What is meant by 4 –12% or 8% SDS-PAGE?

Do the percentage values refer to the percentage of acrylamide in the gel? Yes. The 8% gel is 8 g acrylamide per 100 mL. The “4-12%” gel is a gradient gel, which are useful for separating proteins ...
acvill's user avatar
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2 votes

Need help interpreting Western blot data

Answers to each question: Approximately, yes. In this case, phosphorylated Akt (p-Akt) is supposed to be the active form of the enzyme that goes around the cell doing its job, whereas non-...
Maximilian Press's user avatar
2 votes

AMPK, PAN-AMPK, western blot

Adenosine monophosphate activated protein kinase or AMPK is not a single protein, but is a trimeric enzyme composed of $\alpha$, $\beta$, and $\gamma$ subunits. There are 2 alpha genes, 2 beta genes, ...
MattDMo's user avatar
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2 votes

Preparing sample for SDS PAGE

Usually you would want to keep the amount same, not the concentration. However, if you still want the concentration to be the same then you can add suitable amounts of PBS or your lysis buffer. For ...
WYSIWYG's user avatar
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2 votes
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Optimal pH of protein buffer? Basic principles to adjust buffers according method and analysis

The correct pH depends on the properties of the proteins that you are trying to exploit. These are a good deal different between extracting proteins from tissue and performing Western blots. ...
EdM's user avatar
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2 votes
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Why is it good practice to prepare western blot samples in pairs when you have two experimental groups?

This point is not unique to Western Blot, but to any case-control study: if there is variability inherent in the preparation procedure, this variability will be reduced when the two samples are ...
Roger V.'s user avatar
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2 votes
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When analysing phosphoproteins via Western blot, why is total protein level of the target protein recommended as an internal loading control?

It is because you don't know how your treatments affect the expression of the total specific protein; perhaps it down- or up-regulates the amount of protein being made, but the relative proportion of ...
bob1's user avatar
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2 votes
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In enhanced chemiluminescence in western blots, will the horseradish peroxidase eventually get used up?

The HRP is an enzyme so it isn't a component of the reaction. By definition, enzymes only catalyze reactions but aren't an actual component of the reaction. As such they don't get used up during the ...
yp66t89's user avatar
  • 226
2 votes
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Can Western Blots be used to quantify the activity of a protein?

I see 3 parts to a complete answer: If you define the phosphorylated protein as active, and non-phosphorylated protein as inactive, and you have a total protein blot (as you say, via pan-specific ...
S Pr's user avatar
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2 votes
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What is background signal in a Western blot?

In this context (and many others), the terms "background signal" and "noise" can almost be used interchangeably, if that helps. Generally speaking, noise or background refers to ...
MikeyC's user avatar
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2 votes
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What for a Kinase Assay?

There's a very simple answer: scale. If a drug company wants to screen a million-compound library to find which ones inhibit the kinase activity of a certain target, they're not about to do a million ...
MattDMo's user avatar
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1 vote

choosing the right housekeeping gene for Western Blotting analysis for liver lysates

Use a total protein stain. Irrespective of tissue type, normalising your immunoblot signal using a total protein stain is a good, probably superior, approach (ref1, ref2, ref3). Many journal ...
Michael_A's user avatar
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1 vote
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What is the best way from remove background signal from a band when doing Western blot image analysis?

I haven't done immunoblots for years, and didn't do much quantitative analysis of them even then, so caveat emptor. However: 1) "Are there any ways to determine how wide you want the band for ...
Maximilian Press's user avatar
1 vote
Accepted

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

Generally no, you can't directly compare them and you can't easily compare across gels. I have done many hundreds of western blots, a large proportion of which were for quantitation, and in general ...
bob1's user avatar
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1 vote
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Is it necessary to calculate lane normalisation factor when doing western blot data analysis?

Generally speaking, the proper way to quantify a western blot is to normalize to a loading control such as Actin or GAPDH. In this case it would be (pGSK3/Actin)/(GSK3/Actin) as total GSK3 is not a ...
yp66t89's user avatar
  • 226
1 vote
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Use of western blotting

They are doing a peptide competition assay (see abcam protocol). The antibody is incubated with a purified sample of the antigen and used in a normal Western-Blot of cell lysates. Any cross-...
aretaon's user avatar
  • 131
1 vote

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

That's exactly it. Triton is a detergent and incorporating it just makes it a little more difficult for everything to bind. Here's how I imagine blocking buffer working: PBS/TBS: buffering pH milk/...
Ema's user avatar
  • 301
1 vote

Western blot transfer issues

The first thing that comes to mind for me is that the transfer seems excessive... isn't histone sub-20 kDa? I have often transferred similar-sized proteins at 4C for 30 min, 30V. In general, ...
Ema's user avatar
  • 301

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