Loading controls and antibody validation for Western blot quantification

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Today’s post is inspired by a conversation I had with a colleague when teaching them SDS-PAGE and Western blotting. Being well versed in qRT-PCR (R) and flow cytometry (R), they were particularly interested in how and why certain loading controls were used when Western blotting, and what other techniques were used to validate loading, changes in protein expression and antibody specificity. After explaining to them, I thought I would share with you all too.

Preparing samples

Loading controls ideally form part of a multi-step process to ensure a standard amount of protein is loaded onto a gel for each sample analysed. The first step upon isolating samples is often to perform a protein assay. The BCA and Bradford assays are two of the most popular assays because they allow for standardization of sample concentrations before beginning the assay. While a great first step, there are several issues with the technique: the main one being assay interference from contaminating substances or those used to treat, prepare or isolate samples. For this reason, other pre-separation standardization techniques should be considered alongside proteins assays, such as working with a constant sample size or cell number.

Once the sample has been separated and transferred an often-overlooked, control is to perform a membrane stain, which I discussed in a previous postPonceau S staining is a great validation technique to check transfer efficiency, but samples should also look broadly similar if equal amounts of sample have been loaded. With today’s modern image acquisition hardware and analysis techniques, it is also possible to perform total-protein (R) based quantification using Ponceau S (R), Coomassie blue (R) and stain-free methods (R).

  • Azure Quick Tip #1: Ponceau S staining is performed after transfer

    It works with both PVDF and nitrocellulose membranes and is also easily washed off it is hard not to recommend it above other techniques.


Loading controls

So, onto loading controls. The main purpose of loading controls in Western blotting is to ensure the lanes in your gel contain the same amount of sample and that when you transferred onto the membrane, it was done equally. To confirm that alterations in protein levels are due to effective “knock-down” or “knock-out” and not general changes in protein expression, loading controls are essential.

Most of us have a favorited which we use rain or shine with beta-actin and GAPDH being perennial choices. However, consideration of tissue type, protein of interest localization and size should always be performed, as there are numerous examples in literature that demonstrate how loading controls can and do vary widely (R,R,R).

Here is an excellent resource which details some example loading controls along with their molecular weight, and cellular localization. Keep in mind it is also always worth reading the recent literature to assess previous studies.

  • Azure Quick Tip #2: By performing all three control steps during sample preparation and analysis Western blot quantification should become a breeze.

    This is especially true if you're using a bioimaging system. These systems open the door to multiplex labelling and allow loading controls and the sample of interest to be visualized simultaneously, and total-protein quantification and correction.

Antibody controls in Western blotting

Let’s move onto antibody controls. In Western blotting this area is often overlooked in relation to other antibody based techniques such as FACS or immuno-histochemistry (IHC).

This is partly due to the separation of proteins during electrophoresis which makes determining antibody specificity much easier. It is always worth considering introducing controls for important validations, particularly for Western blots you want to quantify.

Table 1. Four types of Western blot controls include positive, negative, secondary antibody, and isotype controls

Control typeWhat it is and when to use
Positive controlWe use a lysate made from a cell line or tissue sample that has the protein we are trying to detect to test if the antibodies are working properly.
Negative controlTo make sure a test is accurate, we use a negative control. This is a lysate from a cell line or tissue sample that doesn't have the protein we're detecting. Negative controls check for antibody specificity, while also revealing nonspecific binding and false positives.
Secondary antibodyWe only add the second antibody on the membrane to check for antibody specify. In this step, we are able to look for nonspecific binding and false positive results cause by the secondary antibody.
IsotypeIsotype controls help make sure the primary antibody is specifically binding solely to the constant region (Fc) of the primary antibody. Use this for lysates that may be rich in immune cells, or heavily degraded samples.

Positive and Negative Controls

Positive controls are the most widely used in Western blotting and there are several types available. Full length recombinant proteins make an ideal positive control as they should separate identically to the natural protein and can also be used as standards for further quantification if a standard curve is generated.

However, recombinant proteins are not available for proteins and the cost of generation can be prohibitive. In these instances positive cell or tissue samples can be used, including those which have been modified to over-express the protein of interest.

  • Azure Quick Tip #3: Working in the other direction negative control samples can also be used, while a complete blank can be used a better control is a lysate in which the protein of interest is lacking.

    This can be sourced naturally if the protein of interest is relatively tissue specific, or can be generated by knockdown or knockout.

Secondary-only and Isotype Controls

Secondary-only antibody and isotype controls are a mainstay of IHC and FACS; however they are not widely used in Western blotting. Secondary-only antibody controls are performed, as the name suggests, by omitting the primary antibody step from the protocol. This will determine if any non-specific binding or false positives are arising from the secondary binding directly to proteins or protein fragments.

Isotype controls perform a similar function, in checking for non-specific binding, but rather focus on the binding of the constant region (Fc) of the primary antibody, rather than the paratope. This test is particularly important for lysates which may be rich in immune cells which often feature Fc receptors, or heavily degraded samples where non-specific biding can occur.

So to summarize, there are numerous controls that can be performed to ensure your Western blot is optimized for quantification and that you’re observing truly specific binding. While obviously important in its own right, evidence of proper controls is also of growing interest to journals seeking the best quality, repeatable work. So rather than just doing a quick beta-actin strip and re-probe, you should consider some of these additional techniques as well.


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