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, and what other techniques were used to validate loading, changes in protein expression and antibody specificity.
Sample preparation and loading controls
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 with the BCA and Bradford assays being two of the most popular, allowing standardisation 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 post. Ponceau 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 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). However, as Ponceau S staining is performed after transfer, works with both PVDF and nitrocellulose membranes and is also easily washed off it is hard not to recommend it above other techniques.
So, onto loading controls. Most of us have a favourite which we use rain or shine with beta-actin and GAPDH being perennial choices. However, consideration of tissue type, protein of interest localisation and size should always be performed as there are numerous examples in the literature which demonstrate that 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 localisation, but it is also always worth reading the literature to assess previous studies.
However, by performing all three control steps during sample preparation and analysis Western blot quantification should become a breeze, especially if using a bioimaging system which opens the door to multiplex labelling, allowing loading controls and the sample of interest to be visualized simultaneously, and total-protein quantification and correction.
Antibody controls in Western blotting
Moving on to antibody controls, in Western blotting this area is often overlooked in relation to other antibody based techniques such as FACS or immuno-histochemistry (IHC). In part this is due to the separation of proteins during electrophoresis which makes determining antibody specificity much easier. However, it is always worth considering introducing controls for important validations, particularly for blots which will be quantified.
Positive and Negative
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.
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. Again 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 and isotype controls are a mainstay of IHC and FACS however they are not widely used in Western blotting. Secondary only 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 optimised 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 reprobe as previously you should consider some of these additional techniques as well.