1
Protein Identification Using Western Blotting
The ability to detect and identify proteins in a gel is limited because probes cannot
penetrate the gel very well. We can obtain information such as molecular weight and
relative amounts of protein. If the proteins are transferred to a membrane then they are
easily accessible to probes. The elution of proteins from polyacrylamide gels was first
described in 1979 by Towbin et al. Proteins are transferred (blotted) to a membrane
made of nitrocellulose or other material using an electric current and transfer buffer.
Various types of molecular probes can be applied to the membrane to identify a specific
protein. Many use an immunological detection system to locate specific proteins.
We will use an immunoblot alklaline phosphatase system. Following binding of the
protein to the membrane, the remaining protein binding sites on the membrane surface
are blocked with gelatin or equivalent proteins which will not react with the primary or
secondary antibodies. The membrane with bound antigen is then incubated with first
antibody, specific for the antigen to be detected. The membrane is washed to remove
unbound antibody and incubated with the respective goat anti-rabbit second antibody
which has been conjugated to alkaline phosphatase. A color development reagent
containing 5-bromo-4- chloro-3-indoyl phosphate (BCIP) and nitroblue tetrazolium (NBT)
yields purple bands on a white background.
A typical procedure includes the following steps:
1 .Electrophoresis
2. Pre-transfer equilibration
3. Electrophoretic transfer using nitrocellulose membrane
4. Total protein stain control membrane to monitor transfer efficiency
5. Block unbound membrane sites
6. Incubate with primary antibody
7. Wash away unreacted material
8. Incubate with conjugated second antibody
9. Wash away excess
10. Detect antigen
It is important to select primary and secondary antibodies that are compatible. For
example: in this experiment we are detecting bovine serum albumin in the serum sample.
The primary antibody is a rabbit anti-bovine albumin antibody. The secondary antibody is
part of a goat anti-rabbit IgG (H+L) alkaline phosphatase conjugate. A color reagent then
reacts with the AP conjugate to show the location of the bovine serum albumin on the
membrane. The location on the membrane then can be compared to the Coomassie blue
stained gel to identify which band on the gel is the serum albumin.
2
Procedure for Protein Transfer
1. Cut the nitrocellulose membrane and the filter paper to the dimensions of the gel. One
piece of membrane cut in half will serve two gels. Always wear gloves when handling
membranes to prevent contamination. Equilibrate the gel and soak the membrane, filter
paper, and fiber pads in transfer buffer for 1 hour.
2. Prepare the gel sandwich.
.
 Place the cassette, with the dark side down, on a clean surface.
 Place one pre-wetted fiber pad on the gray side of the cassette.
 Place a .sheet of filter paper on the fiber pad.
 Place the equilibrated gel on the filter paper.*
 Place the pre-wetted membrane on the gel.*
 Complete the sandwich by placing a piece of filter paper on the membrane.*
 Add the last fiber pad.
*Removing any air bubbles which may have formed is very important for
good results. Use a Pasteur pipette to gently roll air bubbles out.
3. Close the cassette firmly, being careful not to move the gel and filter paper sandwich.
Lock the cassette closed with the white latch.
4. Place the cassettes in the module. Add a stir bar to the bottom of the module
5. Add the frozen bio-ice cooling unit. Place in tank and completely fill the tank with
buffer.
6. Put on the lid. Place the module on a stirrer and set the speed as fast as possible to
keep ion distribution even.
7. Plug the cables into the power unit, turn on the unit, and adjust the voltage to 30 volts.
Run the blot overnight.
8. Upon completion of the run, turn off and unplug the power unit; disassemble the
blotting sandwich and carefully remove the membrane for development.
Clean the cell, fiber pads, and cassettes with laboratory detergent and rinse well with
deionized water.
9. Place the membrane between 2 pieces of filter paper and allow to dry. Keep the
membrane safe between the filter paper pieces until the next lab.
3
Procedure for Protein Identification
(All steps at room temperature on a shaker)
1. Place the membrane in blocking solution for 1 hour.
2. Wash with Tris buffer solution with Tween-20 TTBS) for 10 min.
3. Place membrane in first antibody solution and leave overnight.
4. Wash with TTBS for 5 min; repeat.
5. Place membrane in conjugate binding solution for 2 hours.
6. Decant and save the conjugate binding solution. Then wash in three different TTBS
washes for 5 minutes each.
7. Place membrane in color development solution for 1 hour (possibly longer) to get
visible purple bands.
8. Decant and save the color development solution.
9. Wash in deionized water for 5 min. Repeat.
10. Photograph the wet membrane with a digital camera (see Dr. Doe for camera).
Analysis
Compare the results of the Western blot to the Coomassie blue-stained gel to determine
the location of the serum albumin bands on the gel. Using the protein standards on the
gel, calculate the molecular weight of all of the proteins on the gel including the serum
albumin. Also in your discussion, include the general principles of protein electrophoresis
and Western blotting.