This is an instructional video on how to conduct a Bradford assay. The following are all the chemicals and instruments you will need for the experiment. Keep in mind that the Bradford reagent is light-sensitive, so it is important to keep it covered while being used like we have done here with aluminum foil.

The assay can be done in a 96-well plate or in cuvettes depending on your samples, in which case the volumes would differ, so make sure you consult with your lab supervisor prior to doing the assay. Start by labeling your reaction tubes where you will dilute your pritine sample in distilled water.

Count enough tubes for the standard curve samples and your samples in triplicates. The first sample of the calibration standards is the one where you only add water, which is also taken as a blank since you will use it to dilute the remaining standards and samples of interest.

For the standard, a BSA solution is used, which is vortexed prior to pipetting. Add the corresponding volumes to each of your calibration standards. This depends on how you prepare the assay in cuvettes or a 96-well plate, so consult with your supervisor regarding calculations.

Here we prepared calibration standards of 20 to 100 micrograms per milliliter.

After having added the BSA, add distilled water to the calibration standards according to your calculations to achieve the desired dilutions.

Don't forget to prepare the duplicate concentration standards.

This way you can use the average values of both standard sets to obtain a calibration curve.

Once you are finished, proceed with your samples of interest. Here we wanted to measure the concentration in this lysozyme solution.

Depending on the protein source or even the protein itself, it might be necessary to dilute the sample prior to measuring. If the concentration is too high, it might exceed the standard curve, so it would be difficult to calculate it accurately. However, do not dilute by a large factor since this could also lead to a value below the standard curve.

If you have never worked with this protein before, but you have enough of it in terms of amount, it might be a good idea to include different dilutions within the same assay so that you can check which dilution factor is most appropriate for future assays.

Prepare the 96-well plate. Include your initials, the date, proteins of interest, or whatever information is necessary when conducting the assay. If you do not have experience with a multi-well plate, it might be helpful to label the cap and mark what you will add where.

Add your calibration standards one by one. Make sure you know which concentration is added to which well. If the samples have been sitting for a while, it is a good idea to either vortex or pipe it up and down to thoroughly mix the sample.

Next, proceed with pipetting your samples of interest.

Once all of the samples have been transferred to the plate, proceed by adding the Bradford reagent.

You will notice a change in color to blue once it is added to the protein samples. Make sure you mix well by pipetting up and down and immediately start a five-minute incubation time at room temperature

after adding the reagent to the first sample.

After the five-minute incubation time, proceed with measuring the concentrations using a plate reader. Don't forget to remove the cap before inserting the plate. Set the instrument to measure at a wavelength of 595 nanometers.

The following is an example of obtained data and calculations based on the absorbance values. The mean absorbance is calculated for each concentration of BSA based on the replicates. These mean values are used to plot absorbance over the respective concentration of BSA. What we need is the y-equation of this linear increase in order to calculate the protein concentration of the samples of interest.

y corresponds to the absorbance values measured whereas x is the known protein concentration you are solving for. Here we have already shown the calculations done for our protein samples. This way you have managed to successfully conduct a Bradford essay and obtain concentrations of your protein of interest.