Immunoblot Protocol
This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.
1. Materials
Protease inhibitor cocktail (Boehringer Mannheim)
2X SDS sample buffer (Novex)
Pre-casted gels (e.g. Novex)
Molecular weight protein standard (e.g. MultiMark Multi-Colored Standard, Novex)
25X Tris-Glycine transfer buffer (e.g. Novex)
100% methanol
Blocking solution containing casein (e.g., Pierce)
Wash Solution I (for 1L: 1g Ovalbumin (Sigma), 1g Dry milk, 1 ml Tween-20 (Bio-Rad), 100 ml 10X PBS, 900 ml distilled water).
Primary antibody
Horseradish peroxidase-conjugated secondary antibody
Wash Solution II (for 1L: 1 ml Tween-20, 100 ml 10X PBS, 900 ml distilled water)
Luminol and oxidizing reagents (e.g., Pierce or NEN)
2. Methods
A: Staining and Laser Capture Microdissection
Place a pellet of the protease inhibitor cocktail into each of the staining dishes.
Stain the 8-12 µm frozen section via the traditional LCM H&E staining protocol.
TIP: Hematoxylin staining decreases subsequent protein recovery and should be avoided if possible. If hematoxylin is absolutely required for visualization during microdissection then approximately twice as many cells should be utilized.
Microdissect 1,000-2,000 cells from the stained frozen section. Limit time spent microdissecting to 30 min or less per slide to minimize protein degradation.
TIP: For lower abundance proteins, more cells may be required for protein detection.
B: Protein Separation by Polyacrylamide Gel Electrophoresis
On top of the transfer film (containing the microdissected cells), continuously pipet up and down 10-20 µl 2X SDS sample buffer for 2-3 min in order for the proteins to go into solution.
Transfer protein solution to a screw-top tube and boil the sample(s) for 3 min.
Spin briefly to collect any condensed fluid.
Set up pre-casted gel and 1X Tris-Glycine-SDS running buffer.
Load all of the sample buffer containing the protein onto the gel. Include a molecular weight protein standard.
Perform electrophoresis at 110V for 1.5-2 hrs.
C: Electrophoretic Transfer To a Membrane (Nylon, PVDF or Nitrocellulose)
Wash gel in 1X transfer buffer (Novex) for 30 min.
TIP: Some sources do not recommend this step as smaller proteins may diffuse out of the gel.
Set up the electrophoretic transfer. Soak 4-5 sponges and 2 pieces of filter paper in 1X transfer buffer. If using PVDF membrane, briefly soak the membrane in 100% methanol then 1X transfer buffer.
Assemble apparatus from negative electrode to positive electrode as follows: 2 sponges, filter paper, gel, membrane, filter paper, 2-3 sponges.
Perform transfer at 40V for 2 hrs.
D: Primary and Secondary Antibody Incubations
Remove membrane and place in a blocking solution containing casein for 30 min at RT.
Wash 3 x 10 min in Wash Solution I.
Incubate with primary antibody overnight at optimal titer (e.g., 1:1000 in Wash Solution I) while rocking at 4°C.
Wash 3 x 10 min in wash solution I.
Incubate with a horseradish peroxidase-conjugated secondary antibody for 1 hr at optimal titer (e.g., 1:20,000 in Wash Solution II) while rocking at 4°C.
Wash 3 x 10 min in Wash Solution II.
TIP: Alternatively, the secondary antibody may be biotinylated where a streptavidin-peroxidase conjugate binds to the biotin on the secondary antibody, greatly increasing the sensitivity for protein detection.
E: Visualization
For the chemiluminescent reaction, incubate with equal parts luminol and oxidizing reagents (Pierce or NEN, Boston, MA) for 5 min at RT.
Develop by X-OMAT autoradiography per recommendation of the manufacturer.
