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 ...
  • 5,979
6 votes

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

β-actin belongs to a class of genes called housekeeping genes. Their proteins typically perform functions that are required across essentially all cell types (for example, β-actin is part of ...
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5 votes
Accepted

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

From what you say, I can only give a few advices: For the quantification, make sure your sample does not contain any substances which disturb the assay. The Bradford assay is for example sensitive ...
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4 votes
Accepted

Are there any disadvantages to HRP conjugated PRIMARY antibodies

You can compare directly coupled antibodies to the classical indirect primary-secondary systems: Directly coupled antibodies Advantages: quicker workflow as only one antibody is used eliminates the ...
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4 votes
Accepted

Mass spectrometry versus western blotting for validation

It is a common practice to prove a result using an orthogonal technique. Like RNAseq followed by qRT-PCR etc. Western blotting is not a robust technique and cross comparisons are difficult because of ...
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4 votes
Accepted

Western blotting with multiple antibodies

What you can do depends on your proteins: If both proteins (your target protein and the loading control) are seperated far enough, you can detect both of them in the same step by adding both primary ...
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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 ...
  • 15.1k
3 votes
Accepted

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 ...
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3 votes

LCMS/MS versus Western Blot

Western blot, though is a commonly used technique and is relatively simple to do, has some issues: Low throughput: it is difficult to analyse multiple proteins simultaneously Limited cross ...
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3 votes

Western blot alternatives

I think that enzyme-linked immunosorbent assay (or ELISA) is the best way to do so, given you have an antibody against the protein to coat the sample wells with... You could use mice to create ...
  • 3,079
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 ...
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 ...
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2 votes

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

I had a similar problem. In my case, I realized that it is absolutely essential to wash the membrane in TBS without Tween (TBS, not TBST) before you use ECL. If you have any amount of soap (Tween) on ...
2 votes

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

After I had check so many possibilities I finally resolve the problem. It turns out that ph of the tris 1,5M for gel preparation was too many basic...which explain a smear of the proteins into the gel
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2 votes

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

There are stable ubiquitinated proteins in mammalian cell lysates even if active proteosomes exist in cells. First, you might want to make sure that the antibody is applicable to WB. Then, you ...
  • 1,116
2 votes
Accepted

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. ...
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2 votes
Accepted

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 ...
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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-...
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, ...
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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 ...
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2 votes
Accepted

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 ...
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2 votes
Accepted

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 ...
  • 4,561
2 votes
Accepted

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 ...
  • 8,784
2 votes
Accepted

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 ...
  • 3,802
2 votes
Accepted

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 ...
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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 ...
  • 8,784
1 vote
Accepted

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 ...
1 vote
Accepted

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 ...
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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/...
  • 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, ...
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