Lab Protocols
Western Blot
The following protocol provides a guideline for detection of PRL-3 by Western blot. Conditions
for detecting tumor-expressed endogenous PRL-3 protein should be optimized by researchers.
Recombinant protein in transfected HEK293 or COS cells:
Load protein samples derived from recombinant PRL-3 cells (equivalent of 1/20 of cells from a 6-cm plate) in each lane of 12%SDS-PAGE.
Block nitrocellulose filter with transferred protein in 5% milk in PBS and 0.05% Tween-20 (PBS/TW). Incubate overnight at 4 degrees C, or 2 hours at room temperature.
Add primary MAb 01-103-02 at 2 ug/ml diluted in 1%BSA in PBS/TW. Incubate 1 hr at room temperature.
Wash in PSB/TW for 5 min, repeat 5 – 6 times.
Add secondary antibody-HRP conjugate, e.g. goat anti-mouse HRP, diluted in 1%BSA in PBS/TW (e.g. 1:4000 dilution). Incubate 45-60 min at room temp.
Wash in PSB/TW for 5 min, repeat 5 – 6 times.
Add HRP substrate. Exposure 30 second to 1 min, or longer with ECL film.
Endogenous protein in tumor cells:
PRL-3 protein expression level in some cell lines can be very low. In order to improve the
quality of Western blot data, immunoprecipitation (IP) should be conducted prior to running the
Western.
Detergent extract of lysed cells can be used as a starting material (microfuge-spun). Protease
inhibitors should be present in the lysate buffer and during the IP step.
Mix cell extract with 2 ug of anti-PRL-3 MAb. Incubate on ice for 1 - 2 hrs. (Use cell extract
from ~ 5x105 to 5x106 cells, depending on the PRL-3 protein level in the cell line.)
Add 20 ul of Protein-A/G slurry, mix for 10-20 min. Spin down beads and boil in SDS-PAGE
sample buffer.
Immunoprecipitation
IP from cell lysate or tissue extract:
Detergent extract of cells or tissue can be used as starting material (microfuge-spun). Protease
inhibitors should be present in the lysate buffer and during the IP step.
Mix cell extract with 2 ug of anti-PRL-3 01-103-01. Incubate on ice for 1 - 2 hrs. (Use cell
extract from ~ 5x105 to 5x106 cells, depending on the PRL-3 protein level in the cell line or
tumor tissue.)
Add 20 ul of Protein-A/G slurry, mix for 10-20 min. Spin down beads and boil in SDS-PAGE
sample buffer.
Analyze IP sample using 01-103-02 on Western Blot.
ELISA - for serum protein (similar protocol can be used for cellular extracts)
Serum obtained from cancer patients or healthy donors may be diluted to 1:20
Add 100 ul diluted serum samples to microtiter plate coated with polyclonal anti PRL-3
antibody (5 ug/ml) and blocked in Blocking Buffer (1% BSA in TBS, Tris Buffered Saline, Ph 7.4
containing 0.05% Tween20)
Incubate at room temperature for 30 min. Wash off un-bound.
Incubate with primary antibody 01-103-01 (5 ug/ml) at 37o C for 30 min. Wash.
Incubate with secondary anti-mouse conjugate of HRP or Alkaline Phosphatase. Wash. Add
substrate. Measure by using an appropriate plate reader.
IHC
Paraffin sections (4 um thick) should be deparaffinized with xylane and rehydrated in ethanol
Endogenous peroxidase activity should be quenched with 3% hydrogen peroxide solution for 15 minutes
Sections should be blocked with 1% BSA for 1 hour
Primary antibody 01-103-01 at 2.5 ug/mL should be incubated overnight at 4°C
After extensive washing, the sections should be treated with secondary antibody for 20 minutes
The reaction should be visualized with diaminobenzidine at room temperature (For counterstaining, slides should be incubated for 2-5 minutes in hematoxylin)
Sections may be hydrated and mounted
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