Protein Loading Technique

Protein loading technique

Ensuring that the correct amount of sample is added to each gel line is one of the most important steps to achieving good and reliable blots. In general, there are two ways to minimise errors: ensuring that the amounts of protein added to each well are equal and that the amount of starting material is also the same.

Adding equal amounts of starting material

This is the less precise of the two methods, but it can be a simpler solution in some cases.
If a hemocytometer is used to check the cell count alongside verifying the weight of the sample, ensuring that the different samples are close or equal in concentration before starting the experiment can help minimise errors. Furthermore, using different sample qualities like a presence of a specific tracer molecule can be used as a verification alongside the previous method.

Adding equal amounts of protein

Different methods of measuring the total protein content can be used with the Bradford assay being the most common of them all. Protocols are widely available both online and from suppliers.
To begin the assay, the Bradford agent has to be added to the samples that need the protein amount verified, following a series of BGG standards with known concentrations.
The next step includes interpolating the sample's concentration from a standard curve, based on the absorbance of the samples.
When the concentration is obtained, diluting or concentrating can be used to achieve the desired concentration for the experiment.

Using a spectrophotometer measuring at 280nm can also be used as a method of determining the amount of protein present, but its time efficiency comes at the cost of poor specificity. Because this method doesn't require a calibration curve, it is better suited for a single protein instead of lysates or other complex mixtures.