8

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 of each pixel have 256 gradations (or 2^8 values of brightness possible between minimum/blank and maximum/saturated). The program only takes the pixel ...


7

ImageJ doesn't have a feature to remove individual lanes. But that shouldn't be a problem. All you have to do is draw the first lane correctly (I'm referring to size). Then press 1. Now, while the selection is still... selected, click inside it, but not on the number (where the cursor becomes hand), and drag it where you want the next lane. And press 2. And ...


7

Blocking buffer Once the proteins in the gel have been transferred to the nitrocellulose membrane it is necessary to coat the rest of the surface of the membrane with an unrelated protein. This is necessary because all proteins will bind non-specifically to the nitrocellulose. Once the membrane has been blocked the only way that antibiody proteins (the ...


7

Sample preparation for protein gels is not a complex task. Simply mix the appropriate amount of sample buffer with your sample and load it. For a 2x sample buffer use equal amounts of sample and buffer, for 5x sample buffer use 4 parts of sample and 1 part of buffer (for examle 40µl + 10µl). Heat the mixed samples for 5 minutes at 95°C, cool them immediately ...


6

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 the cytoskeleton's microfilaments), and are supposed * to be not affected by experimental conditions. In Western blotting, they are run as a "loading control" ...


5

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 against SDS or DTT. See here for more details. This prevents you from loading a sample without enough protein. Make sure that you really have protein on your gel. ...


4

I do not think that a publication quality blot should have such an artifact, but I was able to find something similar by purposely over blotting (not the same as over exposing) a gel. If you use too much primary, secondary, or developing reagent, you can get your HRP signal to "burn in" a membrane where you get a distinct "negative band." By negative ...


4

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 difference in the avidities/affinities of different antibodies. So comparisons can be made only with one protein-control pair in different conditions. LC-MS ...


4

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 antibodies and both secondary ABs into the buffer at the same time. They will bind only to their specific epitope and you will get nice signals. This is ...


4

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 Western blotting. Part of the reason the company chose to recommend the use of Tris-buffered saline over phosphate-buffered saline based buffers was the clearly-...


4

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 chance of a cross-reacting secondary antibody (possibly less background) possibility to use differently labelled antibodies on the same blot Disadvantages: ...


3

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 comparability: since antibodies to different proteins can have different affinities, they cannot be compared with each other. Low sensitivity Not very quantitative LCMS ...


3

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 polyclonal antibodies against your protein by injecting the protein and collecting the serum and purifying it...as long as there isn't similar proteins to the ...


3

In my opinion you should use this formula: $$ \frac{\text{log}_2(\text{Iso}_1/\text{Iso}_2)}{\text{log}_2(\text{GAPDH})} $$ This will normalize the relative fold differences between the isoforms with the loading control- GAPDH. Since both numerator and denominator are log transformed they are in comparable domains unlike the formula-2 that you mention in ...


3

There is no evidence that one is better than the other, most likely because it differs from case to case. Neither you, nor your critics, are right. There is a tiny bit of science in a paper on digitizing blots, generalizing from blots of a specific protein (PMID: 19517440), and they use grayscale for no given reason. Come to think, that is the best paper in ...


3

Vinculin! I love our grad-students, I can't believe I didn't think of it last night. Also, awesome chart, even if it's from a company.


3

So after a lot of work in optimization, I thought I would post what worked best for me. This buffer recipe was able to successfully transfer EGFR and insulin from the same lysate, and a clear band for both (large and small protein respectively). 10% SDS-PAGE gels were transferred at 1A for 10 min. High Current Transfer Buffer 48 mM Tris 15 mM HEPPS 1.0 mM ...


3

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 over a large range of sizes. Read more about the uses and formulations of gradient gels here.


2

Well usually in an ECL kit, one of the reagents is the substrate and the other one is hydrogen peroxide, which activates the substrate for breakdown using the HRP on your secondary antibody (Ab) so I genuinely doubt what you are seeing is the result of ratio difference. In order to make that conclusion you need to run your samples twice (on two separate ...


2

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 your membrane it reacts in some way with ECL and each time you should be getting a different pattern of these stains. Washing the membrane in TBS and then ...


2

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


2

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 would ask if your WB system using the antibody works. You could optimize the condition using just 1D SDS-PAGE followed by WB. For the condition for isoelectric ...


2

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. Extracting proteins means keeping them soluble in aqueous solution. Working at the pI of the protein might be the worst choice as the protein then has no net charge, ...


2

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 IPs and a million Western blots. Instead, they'll use recombinant kinase and substrate peptides, with a variety of readout systems to choose from. Other ...


2

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 e.g. if you have two samples with 1mg/ml and 4mg/ml concentrations and you want to load 20μg of total protein, then you can take 20μl of first sample and add ...


2

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, and 3 gamma genes, each coding for closely-related but not identical isoforms. In addition, like most protein kinases (and many other proteins), it is regulated ...


2

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-phosphorylated Akt doesn't have activity that they are interested in. The authors do show that targets of Akt show characteristics of Akt activity when Akt is ...


2

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 any signal that isn't coming from the thing you're trying to detect (typically in reference to how that signal might interfere or be confused with your "true&...


2

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 phosphorylation doesn't change. For example: Imagine you have 4 samples from different treatments of which you have loaded an equal amount of total protein (...


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