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Western Blotting
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Flash content link: https://vlab.amrita.edu/repo/BIOTECH/CEL/western_blotting/index.swf

 

Materials required

 

  • Micropipettes and tips
  • Microfuge tubes and racks
  • Trays with lid
  • Glass pipette
  • Pipette pump
  • Cell scraper
  • Timer/stop watch
  • Cassette
  • Sponge
  • Filter paper
  • Nitrocellulose membrane
  • Forceps
  • Gel scoop
  • Gel Roller
  • Tissue paper
  • Polythene wrap
  • Waste beaker
  • Ice bag
  • 35mm culture plate


Apparatus

 

  • CO2 incubator
  • Cooling centrifuge
  • Centrifuge
  • Refrigerator(4°C)
  • Dry bath incubator
  • Shaker
  • SDS-PAGE Apparatus      
  •  Blotting Apparatus
  • Gel Documentation System

 

Reagents Preparation

 

  • Phosphate Buffered Saline (PBS-10X):  10.9g anhydrous  Na2HPO4,  3.2g anhydrous NaH2PO4,  90g NaCl, 1000ml DH2O, pH- 7.2.1% SDS(10ml): 1% SDS, 10mM EDTA, 50 mM Tris HCL,  pH- 8.1
  • DDH2O : 500ml
  • Sterile Milli-Q water: 200 ml
  • Running Buffer: 25mM Tris base, 109 mM Glycine, 0.1% SDS, pH- 8.3
  • Sample Buffer: (Leammli buffer): 4% SDS, 20% glycerol , 10% 2- mercptoethanol, 0.004% bromophenol blue and 0.1125M tris HCL, pH-6.8
  • DH2O- 250ml
  • Prestained Molecular weight
  • Transfer Buffer Without SDS(10X): 303g Trisbase, 1440g glycine, 10 L DDH2O
  • Transfer Buffer(1X): 50ml of 10X transfer buffer without SDS, 100ml of methanol, 350 ml DDH2O.
  • Ponceau Stain: 1g of ponceau S, 50ml acetic acid and make up to 1L with DDH­2O.
  • 5% Non-Fat Dry Milk Solution: 50g Non-fat dry milk in 1000ml TBS-T.
  • TBS (10X): 24.23 g HCl, 80.06g NaCl,  800 ml DH2O, pH- 7.6  and make up to 1L.
  • TBS-T (1L): 100ml of TBS (10X) , 900 ml of DH2O, 1 ml Tween 20.
  • Primary Antibody (AntiGFP- Antibody)
  • Secondary Antibody (Antimouse –Antibody)
  • 5% BSA: 50g Bovine serum Albumin in 1000ml TBS-T.
  • Alpha Luminal : 200ml
  • Hydrogen Peroxide: 100ml

 


Procedure


Step1: Sample Preparation  

 

  1. Take the transfected control and test plate used in the transfection experiment used in the virtual lab experiment. These plates are the samples. These plates are the samples. Each plate containing EGFR protein.           
  2. Pour off the medium from both of the culture plates into a waste beaker.
  3. Wash cells with PBS solution into the each culture plate.
  4. Carefully remove the PBS solution from both culture plates.
  5. Add 150µlof 1% SDS lysis buffer to each of the culture plate.
  6. Use a cell scraper to scrape cells from the bottom of the plates and mix well to get a homogenate lysate.
  7. Transfer the homogenate lysate to a 1.5ml fresh microfuge tube.
  8. Allow samples to stand for 5 minute at room temperature.
  9. Centrifuge the resulting mixture at 14,000g for 15 minutes at 25°C to separate cell debris from protein.
  10. Transfer supernatant to a new microfuge tube and store at -20°C.

 

 
Step 2: SDS PAGE

 

  1. Determine the total protein concentrate in the sample by testing a small portion of the lysate with a protein quantitation assay  any  like “Construction of Protein Standard Curve  using  Folin’s  Lowry Method “ (shown in  virtual lab biochemistry experiment). This will assist in loading equal sample across wells
  2. Prepare control (C) loading samples by diluting 7.5 µl of control, 4.5 µl  of sterile Milli –Q water and 4 µl of reducing sample buffer.
  3. Prepare test (T) loading samples by diluting 7 µl of test, 5 µl of  sterile Milli –Q water and 4 µl of reducing sample buffer.
  4. Centrifuge the control and test samples  at 10,000 rpm for 1 minute .
  5. Heat samples at 100 °C in a dry bath for 5 minutes.
  6. After heating the samples are again centrifuge at 12,000 rpm for 1 minute.
  7. Prepare the SDS-PAGE gel by inserting it into the electrophoresis apparatus and filling with running buffer. Rinse the wells of the gel with running buffer and verify there are no bubbles.
  8. Load the 10 µl of control and test samples in appropriate well.
  9. Load the 10 µl of prestained molecular weight marker in the first well.For monitoring the separation during electrophoresis, and subsequently variefy the protein sizes during analysis.
  10. Run the gel until the loading buffer reaches the bottom. This is typically 45-60 minutes at 200V.

 

Step 3: Membrane Transfer (wet transfer)

 

  1. Remove the gel from the cassette and float in transfer buffer.
  2. Choose nitrocellulose membrane for transfer. (PVDF membrane needs to first be activated in methanol for 2 minutes). Incubate membrane in c old transfer buffer for 10 minutes.
  3. Set up gel/ membrane sandwich by placing the transfer cassette in cold transfer buffer. Create a sandwitch stack by placing the components such as sponge, filter paper, gel, membrane, filter paper, sponge from the cathode  to the anode. So that the negatively charged proteins moves from the gel into the membrane.
  4. Use a clean roller with layer to gently roll out any bubbles present.
  5. Lock the cassette and place in the transfer apparatus containing cold transfer buffer. In order to prevent heat buildup, it is beneficial to transfer with a cold pack in the apparatus.
  6. Perform the transfer at 100v for 130 minutes.
     

 Step 4: Immunoblotting

 

  1. Remove the membrane from the cassette and wash three times in DDH2O.
  2. Optional step: verify protein transfer by Ponceau staining the membrane or Coomassie staining the gel.
  3. Swirl the tray in order to sink the membrane fully in the stain.
  4. After swirling, the tray with membrane and keep it as such for 1hour in a shaking platform.
  5. Decant the Ponceau staining and wash with DH2O.
  6. Incubate the membrane with 5% non–fat dry milk in TBS-T blocking solution for 1 hour at room temperature in a shaking plat form. Do not use milk when probing with phosphor- specific antibodies.
  7. Decant the blocking solution (5% Fat -Dry milk solution) and wash with TBS-T for 5 minutes for 4 times.
  8. Dilute the primary antibody by adding 5ml of 5% BSA and 5 µl of primary antibody (anti GFP) into the membrane.
  9. Incubate membranes with primary antibody overnight at 4°C on with gently shaking.
  10. After incubation, decant the primary antibody and wash membrane with large volume of TBS-T and vigorous agitation five times for 5 minutes each.
  11. Dilute the secondary antibody by adding 5 ml of non–fat dry milk solution (blocking solution)and 5 µl of secondary antibody (anti- mouse antibody) in to the membrane.
  12. Incubate membranes with secondary antibody for 2 hours at room temperature  with gentle shaking
  13. Decant the secondary antibody and wash membrane with large volumes of TBS-T and vigorous agitation 5 times for 5 minutes.

 

Step 5: Detection

 

  1. Mix equal part of ECL reagents by adding 5 ml of hydrogen peroxide and 4 ml of alpha luminal solution into fresh tray with siliver lid or cover the lid with aluminum foil.
  2. Incubate the membrane for 3-5 minutes with hand shaking.
  3. Decant ECL mixture and use a  tissue paper to wipe off excess solution from the corner of the membrane. Place the membrane in a clear plastic wrap, such as a sheet protector, to prevent drying . Do not let the membrane completely dry out.
  4. Expose the membrane to x-ray film and develop , or use a ECL capable imaging device. Relative band densities can now compaired with commercially available software.
     

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