Western Blot

Reagents and Materials

• Induced cultures of protein-producing bacteria
• Reagents A and S and BSA standards (for protein quantification)
• SDS PAGE Gel (12% separating gel and 4% stacking gel)
• Nitrocellulose
• Western Blot apparatus

Protocol

Sample Collection

1. Take cells out of the incubator and wash them with PBS 3x
2. Lyse the cells (we used three different protocols for this step: Freeze-Thaw Protocol for Protein Samples, Glass Beads Cell Lysis Protocol for Protein Samples)
3. This solution should be gooey once you collect it. Boil your sample at 95°C for 5 minutes and pass it through a 20-200μL pipette. If the lysate is passing freely (i.e. not gooey) then freeze the sample or do protein assay. If the lysate is still gooey boil it again and repeat the steps until it is no longer gooey

Protein Quantification

1. Add 20μL of reagent S to each mL of reagent A that will be necessary for the run. This makes reagent A’ (If the solution precipitates warm the solution and vortex. Do not pipette the solution the precipitate might clog the tip)
2. Prepare standards (use BSA at 0.2mg/mL, 0.5mg/mL, 1.0mg/mL and 1.5 mg/mL)
3. In a 96 well plate put in 5μL of your standards as well as your samples in triplicates. Make sure to map it out first
4. Add 25μL of reagent A’ into each well followed by 200μL of reagent B (Reagent B is light sensitive so make sure you pour out reagent B when you need it and do not leave it on bench top for too long)
5. Let the mixture incubate for 15 minutes at room temperature (If there are bubbles in the wells after the incubation step is over make sure to pop them with a needle. The bubbles interfere with the absorbance reading)
6. Go over to the plate reader and turn it on and let it calibrate, after it is don’t calibrating open the program called “Softmax Pro”. Double click the panel on the right hand side of the window and change the absorbance wavelength to 750 nm. Also make sure you agitate the plate in the plate reader for 1 second before reading. After you generate the data go to “File”, then “Export file” and make sure you save the data as .txt format in your respective folder and email/ put it on a hard drive (Saving as .txt will allow you to copy into excel without any problems)

Sample Preparation

1. To prepare sample add 40-50µg of protein in Laemmli Sample Buffer + B-mercaptoethanol (950µL sample buffer+ 50µL B-mercaptoethanol). The final volume of your sample should be 20μL for each gel i.e, if you’re running multiple gels adjust this to your procedure. Perform this procedure in a fume hood
2. Boil the samples at 96°C for 5 minutes (Do this right before loading. If you do need to store your samples that are prepared with buffer put them on ice (short term), or in the -20oC (long term)
3. Load the sample into your SDS PAGE Gel (12% separating gel and 4% stacking gel). Load 20μL of sample into each well and run the gel at 80V for approximately an hour. Keep an eye on it every 20 minutes
4. Fix gel in fixing solution (50% methanol, 10% glacial acetic acid) for 1h or overnight with gentle agitation
5. Stain gel in staining solution (0.1% Coomassie Brilliant Blue R-250, 50% MeOH, 10% glacial acetic acid) for 20 minutes with agitation
6. Destain gel in destaining solution (40% MeOH and 10% glacial acetic acid). Change the solution several times until the gel is destained
7. Store the gel in 5% acetic acid if necessary

Transfer (using the BioRad trans turbo machine

1. Cut nitrocellulose, 6 pieces of filter paper/ gel into the correct size pieces
2. Soak the nitrocellulose, the filter paper and the SDS-PAGE gel in transfer buffer for 10 minutes (soaking the gel will allow the transfer to actually happen)
3. Assemble the transfer apparatus as per instruction in the manual (the proteins travel from negative to positive i.e top cassette to the bottom. Make sure the membrane is closer to the bottom than the gel)
4. Run the blotting machine at 1.3A, 25V for 45- 60 minutes to get effective transfer

Ponceau staining (optional)

1. Mix the powdered ponceau S into 5% acetic acid at a concentration 0.02% (w/v)
2. Incubate the membrane at room temperature for 5 minutes and then wash with TBST until the buffer runs clean, i.e not red/pink.

Primary Ab staining

1. Dilute antibody in appropriate concentration in 5% skim milk and 0.05% sodium azide.
2. Incubate the membrane with the antibody (anti his 1:1000) from Qiagen at 4°C overnight / over the weekend (store the antibody after use at 4°C and you can reuse it).
3. Wash your membrane with TBST three times for 10 minutes

Secondary Ab staining

1. Dilute anti-mouse antibody (1:20000 from Bio-Rad) in 5% skim milk and no sodium azide (make this fresh every time and do not store)
2. Incubate at room temperature for 1 hour
3. Wash the membrane with TBST three times for 10 minutes

Development

1. Add the two ECL components 1 and 2 in 1:1 ratio approximately on a piece of saran wrap and cover up your membrane with saran wrap
2. Image using film