Lab Methods: Protein Testing

Method to Test Intracellular Protein

Background:

All cells are made of a few basic building blocks:

  1. A lipid membrane provides a simple shell
  2. Water fills the internal space and provides a working fluid
  3. Carbohydrates store and provide energy and can hold the cell structure
  4. Proteins provide energy, structure, and function

Proteins are the functional units within a cell. They can be used for building internal cell support (fibrils) or external structures (hair/keratin). Active proteins can be transporters (ion channels to move sugar and salts), motors (muscle contraction/myosin), enzyme/catalysis (sugar hydrolysis/cellulase), or builders (DNA replication/polymerase). Signal proteins can be used to identify foreign items (immune response and antigen) or to regulate activity within the cell.  In any case, the protein content and composition is always changing in response to the demands of the cell. If we assume the cells always have about the same level of activity, the protein content is about constant. So, we can use the internal protein amount as a proxy for the actual cell concentration. Here we measure the internal cellular protein content

How this test works:

The centrifuge spins the vial to create a centrifugal force and press the cells into the bottom of the tube. We remove the liquid that might contain extra-cellular proteins (typically enzymes created for some other external function or growth medium compounds that might interfere with the test). A little sodium hydroxide and heat will break open the cell and release the proteins into the liquid. The proteins in the liquid then react with the Coomassie Brilliant Blue G-250 dye to cause a color change. This color change is measured as optical density. We can correlate the color change to the amount of a known protein (usually bovine serum albumin). The protein amount can be correlated to a real dry weight measurement of the cells (which is determined some time earlier for the specific cell type.) This test provides an indirect method of cell measurement. When combined with other information (dry weight data, metabolic activity, etc.) it can give a relative level of protein content in the cell during different growth stages.

 

  1. Obtain a 1.0 mL cell sample (well mixed)
  2. Put the sample into a micro centrifuge tube.
Flasks with T. reesei for testing.

Flasks with T. reesei for testing.

Microcentrifuge tubes in a pile

Previous samples were frozen in microcentrifuge tubes and need sorted.

Samples in microcentrifuge tubes are sorted.

Samples in microcentrifuge tubes are sorted.

 

 

 

 

 

 

 

  1. Centrifuge to pack the cells into a pellet, use 8,000 to 10,000 rpm for 10 min.
  2. Discard the liquid.
  3. Add 1.0 mL of 0.2 M NaOH.
  4. Vigorously mix to re-suspend the cells.
  5. Heat the cells and sodium hydroxide in a sealed tube in hot sand for 20 minutes at 100°C.

 

Microcentrifuge tubes are loaded into a small centrifuge

Microcentrifuge tubes are loaded into a small centrifuge

Sodium hydroxide solution is added to each cell pellet to disrupt the cell membrane.

Sodium hydroxide solution is added to each cell pellet to disrupt the cell membrane.

Closed microcentrifuge tubes are heated to release the proteins.

Closed microcentrifuge tubes are heated to release the proteins.

 

 

 

 

 

 

 

  1. Cool the tube and remove exterior sand.
  2. Centrifuge again to pack cell debris.
  3. Carefully remove a fraction of the cell-free protein containing liquid from the tube (10 uL) and put into a testing vessel (microplate well).

 

Each tube is ordered and opened.

Each tube is ordered and opened.

Each sample is pipetted into a well in a microplate (96 wells per plate)

Each sample is pipetted into a well in a microplate (96 wells per plate)

 

 

 

 

 

 

 

  1. Add protein reacting dye (Coomassie Brilliant Blue) to the small sub sample (200 uL into the microplate well).
  2. Allow reaction to occur for 10 minutes, not longer than 60 min at room temperature.
  3. Measure the optical absorbance at 595 nm

 

Loading protein dye into the pipette tips (12 at a time)

Loading protein dye into the pipette tips (12 at a time)

Loading protein dye into a microplate, row by row.

Loading protein dye into a microplate, row by row.

Microplate with protein and dye (darker blue equal more protein)

Microplate with protein and dye (darker blue equal more protein)

 

 

 

 

 

 

 

  1. Compare the results to a standard calibration curve.
  2. Use another correlation to convert protein to dry weight.

 

Sort and Convert the raw readings into mg/mL protein

Sort and Convert the raw readings into mg/mL protein

Protein Plot

Protein Plot

Leave a Reply