How to Calculate Antibody Concentration

Written by sarah parrish
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How to Calculate Antibody Concentration
Always take notes in a lab book. An inaccurate map of a multiwell plate can render assay results useless. (Thinkstock Images/Comstock/Getty Images)

One of the most common methods of calculating a protein concentration in a laboratory setting is to use a Bradford Assay. The Bradford Reagent is a colorimetric reagent that changes colour based on protein concentration. By combining a known standard curve with the Bradford Reagent, you can create a measure against which you can calculate the concentration of any protein, including antibodies.

Skill level:
Moderately Challenging

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Things you need

  • Purified antibody in a known buffer
  • Buffer matching the suspension of the antibody
  • Lyophilised bovine serum albumin (BSA)
  • Microcentrifge tubes
  • Laboratory pen
  • Vortexer
  • Laboratory scale
  • Weighing paper
  • 1ml pipette
  • Bradford reagent
  • Multi-well plate
  • Colorimetric plate reader
  • Microsoft Excel
  • Lab book
  • Pen

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Instructions

    Create a Standard

  1. 1

    Measure 200 mg of BSA on weighing paper using a laboratory scale. Enclose it in a microcentrifuge tube. Add 2ml of buffer to the microcentrifuge tube containing the 200 mg of BSA. Label this tube "1."

  2. 2

    Vortex Tube 1 until the BSA is completely dissolved.

  3. 3

    Remove 500 ul of the BSA in buffer and transfer it to a second tube. Label this tube "2." Add 500 ul of plain buffer to Tube 2.

  4. 4

    Remove 200 ul of BSA in buffer from Tube 1 and transfer it to a new tube. Label this tube "3." Add 800 ul of plain buffer to Tube 3.

  5. 5

    Remove 100 ul of BSA in buffer from Tube 1 and transfer it to a new tube. Label this tube "4." Add 900 ul of plain buffer to Tube 4.

  6. 6

    Remove 10 ul of BSA in buffer from Tube 1 and transfer it to a new tube. Label this tube "5." Add 990 ul of plain buffer to Tube 5. Tubes 1- 5 comprise a graded standard series. The first tube has a known concentration of 100 mg/ml; the second, 50 mg/ml; the third, 20 mg/ml, the fourth, 10 mg/ml; and the fifth, 1 mg/ml. Each tube contains approximately 1ml of solution.

    Create Antibody Dilutions

  1. 1

    Add 5 0ul of purified antibody to 95 0ul of buffer. Label this tube "A."

  2. 2

    Add 10 ul of purified antibody to 990 ul of buffer. Label this tube "B."

  3. 3

    Add 2 ul of purified antibody to 998 ul of buffer. Label this tube "C."

    Load the plate

  1. 1

    Place 0.5ml of plain buffer in the first wells in two columns of the multiwell plate.

  2. 2

    Place 0.5ml of the contents of Tube 1 in the second wells of the first two columns in the multiwell plate. Continue down the graded series until the first two columns contain 0.5ml of one BSA standard solution from each of Tubes 1-5, including wells for plain buffer.

  3. 3

    Add 0.5ml of Bradford Reagent to each well. Swirl the plate to mix the reagent with the proteinaceous solutions, being careful not to spill the contents of any of the wells. Wait 5 minutes.

  4. 4

    Read the plate according to the protocol specific to your plate reader at a wavelength of 595 nm.

    Calculate Antibody Concentration

  1. 1

    Copy the values read from the plate reader into Microsoft Excel.

  2. 2

    Create a graph from the two columns of values representing the BSA standard using the Chart Wizard in Microsoft Excel.

  3. 3

    Plot the points measured from the antibody dilutions in the Excel graph. Read the corresponding concentration of protein according to the value of the y-axis for the antibody dilution point on the Excel graph.

  4. 4

    Depending on whether you're using the values obtained from Tube A, B or C, multiply the value by either 20, 40 or 500, respectively. The resulting value is the concentration of your purified antibody.

Tips and warnings

  • Use protein-free suspension buffers or your results will be inaccurate because the Bradford Reagent reacts nonspecifically with protein.

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