13.  Western  Blot  Analysis

A. Principle :

      The Western blot analysis, first described by Towbin et al. (1979) is an extremely   sensitive method for visualizing specific proteins in complex antigenic mixtures. In  this technique, the separated protein components in SDS-PAGE are transferred onto the surface of  nitrocellulose sheet for immobilization and then identified based on their  immunological reaction with antibody and enzyme labelled or radio-iodinated second antibody probe. This technique is useful for comparing reactivity of several sera with a single antigenic preparation or of a single antibody preparation with several antigen sources.  

B. Materials :

a. Transblotting apparatus with power supply and gel cassettes.

b. Nitrocellulose membrane (Schleicher and Schuell, BA 85, 0.45 m pore size)

c. Whatman filter paper No.3

d. Culture tubes with screw caps.

e. Water bath with shaker.

  f. ransfer buffer (Tris glycine buffer, pH 8.3)                               (Appendix-1)

g.   0.25 M SPB, pH 7.2     (Appendix-1)

h.   Quench solution (5% skimmed milk powder in PBS)

Dissolve 10g skimmed milk powder (SMP) in 200 ml PBS

i.    Washing buffer (PBS/T)   (Appendix-I)

  j.    Citrate-Phosphate buffer (pH 5.0)   (Appendix-I)

k.   Diaminobenzidine substrate    (Appendix-I)

l.    Anti-human IgG peroxidase conjugate.

m.  Transbloting buffer     (Appendix-I)

C. Method :  

a)   Pre-cooling of transfer buffer and preparation of gel and nitrocellulose paper (NCP)

      1.   Pour  5  litres  of  transfer  buffer  into the transblotting apparatus tank and keep in cold  atleast 4 hrs before transblotting for precooling.

      2.   After SDS-PAGE run is over, incubate the gel in transfer buffer for 30 minutes and then  change the gel into fresh transfer buffer and incubate for another 30 minutes. This will  eliminate the possible swelling of the gel during transblotting.

      3.   During the second 30 minutes incubation of gel, cut a piece of NCP slightly larger than the gel and wet it by rolling onto distilled water. When completely moist, submerge for  5-10 minutes and then place it in transfer buffer to equilibrate for 15 minutes.

b)   Assembling the cassette

      1.   Pour one litre of transfer buffer into a tray large enough to hold the gel and cassette to a depth of about one inch.

      2.   Place the grid with molded extension in tray so that pins face upwards.

      3.   Place  the  dacron  sponge  on  it  using  the  pins  as  a  guide  and  press carefully on the sponge to avoid any trapped air bubbles.

      4.   Cut off molecular weight standards lane and reference lanes, if  any,  from  the  gel for protein staining. Notch the gel for orientation.

      5.   Cut 4 Whatman 3 filter papers slightly larger than the gel and wet by placing them in Transfer buffer.

      6.   Pick up the gel with two wet filter papers and place on the sponge with the filter paper next to the sponge.

7.   Cover  with  NCP,  dull  side  in contact with gel and mark NCP overhang with pencil to mark the orientation.

      8.   Place a second piece of Whatman 3 filter paper, sponge pad and grid. Recheck the order of layers which should be grid with extension-sponge pad-filter paper-gel - NCP - filter paper-sponge pad-grid without extension.

      9.   Close the cassette by matching the clips in one half with the holes in the other. Slide the top  grid  down  slightly  into  the  grooves  on  the clips until it holds firmly in place, if necessary  rubber  bands  can  be  used  to  apply  additional   pressure  to  the  ends of the cassette.

c)   Transfer

      1.   Place the assembled cassette in the buffer tank with the gel side (i.e. the side with grid having extension) facing the cathode and the NCP side (i.e. the side the grid having no extension) facing the anode.

      2.   Top  up  with  more  transfer  buffer  if  necessary and place the power supply lid on the chamber so that the anode female connector (+) on the lid contacts the electrode which is to function as the anode.

      3.   Turn the Voltage, adjust knob fully clockwise. The current will start at 0.6 to 0.8 amps and

run for one and half hours. The current will increase to about 1.2 amps during the run  ( if  the  transfer  buffer  gets  hot during the run, decrease the  voltage and increase the time).

      4.   After the electrophoretic transfer is completed, turn the voltage, adjust knob fully  counterclockwise. Turn off the power and unplug the lid from the main. Carefully lift the power supply lid of the buffer chamber. Place the cassette in an empty tray with gel side down.

Disengage the two halves and remove the sponge and filter paper. Very carefully            remove the NCP from the gel by starting at one end and "Peeling" it away.

      5.   Place the NCP (Dull-side up) in Quench for 2 hr at 370C or overnight at 40C.

      6.   Stain the gel with coomassie blue to assess the transfer.

      7.   After quench is complete, wash NCP four times, each time for 15 minutes with PBS/T with 2% SMP.

      8.   Cut NCP into strips for immunoblotting and identify the strips with pencil.

d)   Immunoblotting

1.       Incubate NCP strips in optimally diluted sera (1:50) or antibody solution and controls in PBS/T with 0.5% BSA at 370C for 2 hrs (in screw cap culture tubes).

2.       Wash NCP strips in PBS/T four times, each time for 20 minutes.

3.       Incubate the strips with optimally diluted peroxidase labelled second antibody at 370 C for 2 hrs.

4.       Wash the NCP strips in PBS/T as described above.

5.   Place the NCP strips  in  freshly  prepared  DAB-substrate solution in culture tubes or petridish for about 30 minutes in reduced light.

      6.   Stop the reaction by transferring the strips to water, air dry and preserve.

Note :

      1.   The dull side of NCP should be up in all wash steps to protect from abrasion, since it will

            receive the blotted antigens.

      2.   Avoid air bubbles in all stages of assembling the cassette.

      3.   Transblotting should preferably be done at 40C. It can also be done at low voltage and  current for long hrs (30 V and 0.1 Amp for 24 hr).

      4.   During transblotting, buffer circulation is generally not necessary. However if desired, place a magnetic stir-bar in the bottom of tank and carefully place the unit on magnetic stirrer but the turbulance should be maintained at minimum.

      5.   If necessary, all or part of NCP after transblotting can be stored for future use (at   least for 5 days ). The  NCP  should  be  incubated  in  PBS  with 0.01% sodium azide (after Quenching )  for  30  minutes,  dried,  sealed  in  a  plastic  bag  and then  stored at 40C.

Remember to wash thoroughly before using to remove azide if peroxidase is used in the immunoblotting.

      6.   Diaminobenzidine  is  carcinogenic  and  so  use gloves while handling and avoid breathing.

It is also light sensitive and should be handled in subdued light.

      7.   If   necessary,  stain  the  NCP after transfer with 'ponceau' stain  ( 2.0%  ponceau S in 3% trichloroacetic  acid  in  distilled  water )  for  5 minutes and destain in distilled water to assess the transfer of proteins. After ponceau staining the NCP can be further used for immunoblotting after quenching.

      8.   If desired, after transblotting a part of the NCP can also be stained directly using 0.2%  coomassie  blue  R - 250,  50 %  methanol,  10%  acetic  acid for 5 minutes. It should be destained quickly in 90% methanol and 2% acetic acid to avoid  disintegration of NCP.

      9.   To keep the transblotting unit in good operating order, rinse the chamber and cassettes with  distilled  water  and  dry immediately after the transblotting. Also clean the power supply lid with a clean cloth and keep dry. Do not use strong detergent for cleaning.

References

      1.   Towbin ,  H .  Slahelin ,  T .  and  Bordon,  J.  Electrophoretic transfer of proteins from polyacrylamide  gels  to  nitro-cellulose  sheets  procedure and some applications. Proc.Nat. Acad. SC., USA, 76 (1979), 4350.

      2.   Genes  and  Antigens  of  parasites - A lab. Manual edited by C. LM. Moral, publishing by            UNDP /  Word  Bank /  WHO special programme for Research and Training in Tropical         Diseases.