BCA Protein Assay

BCA Protein Assay


This video will demonstrate how to perform
a BCA protein assay. Here is the equipment you will need. Label the microfuge tubes before you start. You will need to dilute your samples to at
least 2 different concentrations to ensure one of them will fall within the standard
curve. Base on your pre-lab calculation targeting
to make 100 microliters of each standard solution, first pipette the appropriate amount of ddwater
into the microfuge tube, then add the proper volume of protein stock solution. Vortex the microfuge tube to mix the solution. Prepare all your standards and samples. Pipette 10 microliters of your standard solution
and samples in triplicates into the microplate. Either print the microplate
template that is provided on Connect, or draw the template on your notebook. Record the location of each standard and sample
within the well. It is generally easier if you put all standards
in the same columns, that is, A1 – 3 for the blank, B1 – 3 for the lowest concentration,
then C1 – 3 for the next, and so on. Start from the least concentrated standard
to the most so you only need to use one pipette tip. For your samples, you should switch a tip
for each dilution. Try to prevent having any bubbles in the well
as it may affect your absorbance reading. Next, pipette the proper amount of BCA reagent
A into the falcon tube, then add the proper amount of BCA reagent B and mix to produce
the BCA working reagent. Be aware that each well needs 200 microliters
of the BCA working reagent, so make sure you prepare enough working reagent for the assay. It is recommended that you always prepare
an additional 1 ml of working reagent just in case. Mix the BCA working reagent and add 200 microliters to each well. Move the plate in this way to gently mix the
solution. Cover the plate with aluminum foil and place
it in a 37 Celsius degree incubator for 30 minutes. After incubation, take the microplate out
and cool to room temperature. Set up the TECAN spectrophotometer to read
the absorbance of your sample at 570 nm. Remove the foil cover, make sure the well
A1 is at the left top corner when you place the microplate into the plate reader. Save the results in excel file with session#
and group# on the computer as well as on your USB for further analysis. Rinse the microplate with tap water in the
sink then dispose in the garbage bin.

Comments

(1 Comment)

  • Norton Silva

    Thank you!

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