ELISA for BDNF Cort

Purpose:

ELISA is a technique used to probe for specific proteins in tissue lysates.

 

Reagents and Equipment:

  • 96-well microtiter plate (Nunc Maxisorp; VWR, West Chester, PA)
  • Monoclonal mouse anti-human BDNF (clone 35928.11; Calbiochem, San Diego, CA) (249.00)
  • Filter-sterilized PBS (10 pack -61.50)
  • TBS + 5% tween (TBST),
  • 3% BSA in PBST
  • 1M HCl
  • 1M NaOH
  • Filter-sterilized buffer (1% BSA in PBST).
  • Homodimeric recombinant BDNF (rBDNF; Peprotech, Rocky Hill, NJ) (10ug -215.00)
  • 100 μl of polyclonal chicken anti-human BDNF (2.5 μg/ml; Promega) – (403.00)
  • Anti-chicken IgY-HRP (1 μg/ml; Promega)- (139.00)
  • TMB (tetramethylbenzidine; Promega)- (107.00)

Protocol: 

Pouring Gels

  1. Depending on the molecular weight of the proteins of interest, the percentage of acrylamide in the gels will change.  Proteins less than approx. 150kDa will transfer adequately from a 10% gel.  Proteins >150kDa may transfer better from gels < 10%.  See Page 6 for gel recipes.
  2. Assemble the glass plates in the clamps and stand and check for leaks by pipetting water between the plates.
  3. Remove XS water from between the plates with thin blotting paper.
  4. Prepare the separating gel at the desired % acrylamide and pour between the plates to the correct height.
  5. Layer water saturated butanol on top of the gel to prevent drying.  Let the gel polymerize.
  6. Pour off the butanol and rinse with water.  Remove XS water with blotting paper.
  7. Prepare the stacking gel and pour to the desired height on top of the separating gel.  Insert the comb and allow this to polymerize.

Preparing Samples

  1. Thaw the Molecular Weight Markers and 5X Reducing Sample Buffer
  2. Combine 4 parts of sample with 1 part 5X Reducing Sample Buffer and mix well.
  3. Heat diluted samples and Molecular Weight Markers at 95°C for a minimum of 5 minutes.

Assembling the Gels

  1. Once the stacking gel has polymerized, remove the combs and assemble the gels into the electrode assemblies.  Insert the electrode assemblies + gels into the electrophoresis tank.  If only one gel is being run, place an empty gel clamp into the other side of the electrode assembly.
  2. Fill the outer and inner chambers with SDS-PAGE Running Buffer.
  3. Using a syringe filled with SDS-PAGE Running Buffer rinse out the wells of the stacking gel by squirting the buffer into the wells.

Loading Molecular Weight Markers and Samples

  1. Using gel loading pipet tips, load the required amount of molecular weight markers and standards in designated wells.  Use the Western Blot Template on page 5 to keep track of sample placement and blot conditions.
  2. If a single gel is being used for more than one antibody blot, it is convenient to leave a blank well between sets so there is plenty of room to cut the blot later without cutting through samples.
  3. Ensure that enough Molecular Weight Marker is loaded (protein-wise) so that all bands can be visualized with Ponceau S after transfer.
  4. Avoid pipeting bubbles while adding samples to wells as this may cause samples to bubble out of the wells.

Running the Gel

  1. Once all samples and molecular weight markers are loaded, place the electrophoresis chamber lid on and connect to a power supply.
  2. A guideline is to run the gel at 90v for 10 minutes through the stacking gel and then at 100-200v through the separating gel.  Stop applying voltage once the dye front is near the bottom of the gel.

Transferring Proteins from the Gel to the Nitrocellulose Membrane

  1. Pre-wet the blot sponges, filter paper and nitrocellulose/pvdf paper in 4°C Transfer Buffer.  **Never handle nitrocellulose/pvdf membrane with bare hands or even gloves.  The preferred method is to use tweezers.
  2. Remove the gels from the electrode assemblies one at a time being careful not to tear them.
  3. Assemble the blot sandwiches one at a time as shown in Figure 1.

Figure 1-kindly provided by A. Titterness

 

  1. Insert the blot sandwiches into the blot electrode assembly.
  2. Fill the electrophoresis tank with cold Transfer Buffer and add a stir bar.
  3. Perform the transfer on a stir plate in the refrigerator.  Run at 40v for overnight.
  4. After transfer is completed, remove the blot sandwiches and take them apart.  Stain the nitrocellulose membrane with Ponceau S for a few minutes.
  5. When the molecular weight markers show up, mark them with ballpoint pen.
  6. Remove the Ponceau S stain by washing the blot a few times in PBS/Tween-20 wash buffer.
  7. After washing, incubate the blots in 5% Blotto Blocking Buffer for 1 hour at RT or overnight in the refrigerator with agitation.
  8. If desired, the SDS-PAGE gel can be stained with coomassie blue to determine the success of the transfer.  You will expect to see some protein remaining in the gel but it should be quite faint.
  9. Incubate gel with Coomassie Blue in a staining tray for 15 min then pour off the stain back into the stock bottle (it can be re-used).
  10. Destain the gel by filling the staining tray with water and a rolled up large kimwipe.  Microwave for approximately 1-2 minutes…it should get very hot…close to boiling.
  11. Remove from the microwave and put on the Belly Dancer for 5-10 minutes.
  12. Pour off the old water and add fresh.
  13. Repeat steps 13 to 15 a few times until it has been destained sufficiently.

Probing the Blot with Antibodies

  1. Prepare the primary antibody at dilutions recommended by the vendor at dilutions tested in-house and found to be successful.  Dilutions are made in 5% BSA in PBST.  In general, the volume required for an entire 7.5 x 10cm piece of nitrocellulose is approximately 8-10mL.
  2. If necessary, the nitrocellulose can be cut into sections for incubation with different antibodies.  A razor blade on a hard surface works well for this.  Try and be quick so that the blot doesn’t dry out.  It is important to mark each section with ballpoint pen to keep track of what antibody is used on what section
  3. Wash the membrane 3X5 min in PBST on the belly dancer.
  4. Prepare the antibody incubation in a 15mL tube or box.
  5. Lay each piece of nitrocellulose/pvdf into the box/tube. Incubate with the primary antibody for 1-3 hour at room temp on the belly dancer.
  6. Wash blots in PBS/Tween-20 Wash Buffer 3 x 5 min on the Belly Dancer.
  7. Prepare the secondary antibody dilutions in 5%Blotto Blocking Buffer.
  8. When the blots are washed, put them into a box or 15mL tube and add the secondary antibody. Let these incubate for 1 hour at room temp on the belly dancer.
  9. Wash again as in step 6.
  10. Lay each blot onto the parafilm covered surface and pour the TMB substrate over top.  Watch for color development and stop by placing blot into PBS/Tween-20 wash buffer or water.

Scanning the Blot

  1. Turn on the BioRad Gel Doc Imaging System and open up the Quantity One Software.
  2. For imaging blots, use the UV to White light converter screen and the epi-white light source.
  3. Acquire image and annotate to mark the molecular weight markers and lanes if desired.
  4. Save image and print a copy for your notebook.