Western Blot Protocol

Materials Provided

• transfer buffer

• methanol

• filter paper

• TBS: essential for washing, blocking, and antibody incubation

• 5% Tween-20: reduces non-specific antibody binding

• 25% Milk powder: blocking agent

• PVDF membrane

• primary antibody at 1:1000

• colorimetric reagent

• protein ladder

Transfer: Step 1

• wet PVDF membrane, cut to gel size in 100% methanol

• soak in transfer buffer for 30 mins

Transfer: Step 2

• soak SDS-PAGE gel in transfer buffer for 5 mins

• discard the buffer

• repeat this step 2 more times (total 3 buffer changes)

Transfer: Step 3

• soak 2 pieces of thick filter paper in transfer buffer

Transfer: Step 4

• assemble the transfer stack on the platinum transfer apparatus anode, using a test tube or similar to roll out bubble between layers

• the stack (bottom to top): thick filter paper, PVDF membrane, acrylamide gel, thick filter paper

Transfer: Step 5

• replace the cathode and safety cover

• run at 25V and 0.25A for 40 mins

Blocking and Probing of Membrane: Step 1

• place the membrane into suitable container

• ensuring the protein side is up, block non-specific sites by soaking in 20ml of 5% Milk powder in TBS-T for 60 mins

• place the container on the shaking platform, ensure that the membrane is covered in solution

Blocking and Probing of Membrane: Step 2

• remove the blocking solution (dispose in sink) and incubate with 15ml of primary antibody diluted to 1:1000 in 1% Milk powder/TBS-T for 30 min at room temp

Blocking and Probing of Membrane: Step 3

• prepare 50ml of TBS-T (TBS w/ 0.1% Tween20)

Blocking and Probing of Membrane: Step 4

• remove the antibody solution and wash 3 times with 15ml of TBS-T (1×1min, 2×5min)

• each wash can be disposed of in the sink

Blocking and Probing of Membrane: Step 5

• remove the membrane using forceps and place it protein side up on a plastic sheet

• pipette 2ml of colorimetric reagent onto the membrane and incubate until color reaches desired intensity

Blocking and Probing of Membrane: Step 6

• take a photo of the membrane of transilluminator

Fold purification

• measures how much a protein has been enriched after a purification step compared to the initial mixture

• calculated by determining the fraction of a protein of interest in the sample before the purification and comparing it to the fraction after purification

  • i.e the ratio of the purified protein in the sample after purification divided by the ratio of our protein in the sample before purification

• requires:

  • protein concentrations from Bradford assay for the cleared lysate (before purification), and purified protein (after)

  • densitometry data on the Western blot image

  • the volume of the samples loaded on the gel

Fold purification: Example Calculation Data

• second lane = cleared lysate, third lane = purified protein

• band of interest has a molecular weight of 104/102 kDA, which is close to the expected mass of 97kDA

  • therefore, pretty confident this band is correct

• raw volume: lysate = 882, purified protein = 1084

  • therefore 1084/882 = 1.2 x as much recombinant protein in purified lane as in cleared lysate lane

• the SDS-PAGE were loaded w/ 10µl of purified protein and ~3.8µl cleared lysate (3.8µl + 1.2 µl loading buffer)

Fold purification: Calculate Bradford Assay (this is now using our data lol)

Fold purification: Actual Western Blot Loading Volumes

Fold purification

Percent Recovery

• the yield, the percentage of total protein (or activity) you keep after purification step

• requires:

  • total physical volumes of the purified protein and the cleared lysate solutions recovered (not just what was loaded on the gel)

Theory

• detects/quantifies individual proteins within the mix

• first step: protein components are separated based on molecular weight by electrophoresis in SDS-PAGE

• proteins are then transferred to immobile substrate; the transfer process uses the same principle as SDS-PAGE, but the electric current is applied 90º to the gel and the proteins are migrated out of the gel onto the membrane

• prior to incubation with the antibody solution, the membrane is blocked to reduce non specific (non-epitope) protein interactions between the membrane and antibody (we used non-fat dry milk)

  • small amount of detergent in the blocking and wash buffers also help to prevent non-specific binding

Primary Antibody

• specific for protein of interest

• incubated with the membrane in the blocking solution; at appropriate concentrations, the primary antibody should not bind any other proteins on the membrane (it frequently does tho)

Secondary Antibody

• after washing the membrane in buffer/detergent solution to remove unbound primary antibody, a secondary antibody is incubated with the membrane

• this antibody binds to one or more epitopes of the primary antibody and is typically linked to an enzyme that allows for visual identification by producing fluorescence or color change

• the unbound secondary antibodies are washed away and the enzyme substrate is incubated with the membrane so that the positions of membrane-bound secondary antibodies can be detected

• bands corresponding to detected protein of interest will appear and band densities in diff lanes can be compared

What is the difference between primary and secondary antibodies?

Primary:

• directly binds to the target antigen (protein of interest), provides specificity, usually unlabeled

Secondary:

• binds to the primary antibody (not the antigen)

• conjugated to a detectable label (eg. enzyme, fluorophore)

• provides signal amplification (multiple secondary antibodies can bind one primary)