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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). Most of us have a favourite which we use rain or shine with beta-actin and GAPDH being perennial choices. 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. 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). 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. 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. 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. 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. 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. 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. Protein and Western Blotting Reagents and Accessories.
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