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College of Life Sciences
Microbiology and Molecular Biology

Protein Harvest and BCA

September 07, 2026 01:38 PM

Protein Harvest

  1. In hood, aspirate media from wells
  2. PBS wash: gently pipette 1ml PBS over wells and aspirate out again
  3. Add 200μl trypsin and let sit for 5 minutes in the incubator
  4. Quench trypsin with 800μl media 
  5. Out of the hood, pipette up and down to detach cells from base of well and then transfer each well’s content to a labeled microcentrifuge tube
  6. Spin at 900 RCF for 5 minutes
  7. Aspirate down to 100μl
  8. Resuspend in 1ml PBS (wash one)
  9. Spin at 900 RCF for 5 minutes
  10. Aspirate down to 100μl 
  11. Resuspend in 1ml PBS (wash two)
  12. Spin at 900 RCF for 5 minutes
  13. Aspirate supernatant and add 1μl of protease inhibitor then 100μl RIPA to each sample (alternatively mix the amount of protease inhibitor (1μl per sample) and RIPA (100μl per sample) together and add 101μl of the solution to each sample)
  14. Sit on ice for 10-30 minutes
  15. Store at -20 celsius

BCA

  1. Thaw samples 

  2. Sonicate

    1. Setting: pulse 10s at 30%
    2. While off, place probe in sample and keep submerged during pulse
    3. Remove sample from probe while sonicator is off (if you move it to the surface of the sample while it is on it will foam up)

  1. Spin at high speed in the cold room for 10 min
  2. Add 10μl standards in duplicate to a 96 well plate
  3. Dilute samples by 10 (4μl sample in 36μl molecular grade water) and add 10μl of diluted samples in triplicate to the 96 well plate (vortex dilution before adding it to the well plate or protein will settle and you will not get a consistent concentration read in each replicate)

  4. BCA reagent make up:

    1. BCA solution made up in 1:50 ratio of reagent B: reagent A

    2. [(number of samples x 3) + (number of standards x 2)] x 190 = total BCA reagent

      1. Add ~2 more samples to calculation to make sure you have enough
      2. Number of standards = 9
    3. Total reagent / 51 = volume B reagent needed
    4. Total reagent - volume B = volume A reagent needed
  5. Mix up BCA solution in plastic reservoir
  6. Add 190μl of BCA solution to each well with multichannel
  7. Sit for 1hr in non-sterile incubator
  8. Image with plate reader @ 562 nm (use existing BCA protocol on machine)

BCA analysis

Example results:

Yellow = standards

Blue = samples

(See attached link for images)

Set up the following table:

Standards: A through I

Concentrations (in mg/ml): known concentrations of samples as stated

Average: average readings for each standard duplicate

Sample #: any identifying information about each sample for tracking purposes

Average: average readings for each sample triplicate

Interpolated: mg/ml concentration of each sample based on an interpolation of a standard curve done through PRISM

Normalized: divide lowest concentration by sample concentration (in this example it is 253.21/each sample)

μl protein: multiply by 10

μl water: subtract μl protein from 10

(See attached link for example image)

https://docs.google.com/document/d/1oVkHw8lsywXUas9QU7Ij3-y7biwm4aM5K5c75__UXcA/edit?usp=sharing