NanoPro™ Assay
Phospholipase C Gamma 1 (PLCγ1)
SUMMARY
Primary Antibody: Anti-PLCγ1 (Cell Signaling Technology, cat# 2822), anti-phospho PLCγ1 (Abcam, cat# ab53125)
Detection Antibody: Anti-Rabbit HRP (Cell Biosciences, p/n 040-656)
Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) to produce the metabolite second
messenger molecules inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG). Increase of IP3 results in elevated intracellular free
Ca2+. PLC’s are activated through G-protein coupled receptor stimulation as well as tyrosine receptor kinase activation and therefore
bridge both important signaling pathways. The family of PLC's consists of 12 isoforms with different roles in signaling. For example, PLCγ1
forms a complex with activated EGF receptors, which leads to the phosphorylation of PLCγ at Tyr771, 783 and 1245. Here we detect
the phosphorylation of PLCγ1 in HEK293 cells in response to EGF treatment.
RESULTS
EGF treatment results in increased PLCγ1 phosphorylation in HEK293 cells.
HEK293 cells were treated +/- 50 ng/mL EGF for 15 minutes. EGF treatment resulted in a dramatic increase in a pI 5.8 peak detected with
the anti-phospho (Y783) PLCγ1 antibody (right panel, lower trace). Additionally, a slight shoulder in the total PLCγ1 peak profile near pI 5.8
was detected with the anti-PLCγ1 (total) antibody (right panel, upper trace). Similar peak profiles have been generated in EGF treated HeLa
cells and serum-treated U937 cells (data not shown).
PROTOCOL
Cell Preparation
Cell culture:
HEK293 cells (ATCC, cat# CRL-1573) were cultured in EMEM (ATCC, cat# 30-2003) containing 10% FBS (Irvine Scientific,
cat# 3000-A) and 1x Penicillin/Streptomycin/Glutamine (JRS Scientific, cat# 20020). Cells were split 1:5 every 3 days using
0.25% Trypsin (Cellgro, cat# 25-053-CI) at 37°C for 3-5 minutes. Data shown from cells at passage 5.
Pre-treatment:
Before EGF stimulation, cells were placed at 37°C, 5% CO
2 overnight in starvation medium containing MEM without serum.
Treatment:
50 ng/mL EGF in MEM without serum for 15 minutes at 37°C, 5% CO
2.
Lysis buffer:
Bicine/CHAPS Lysis Buffer (Cell Biosciences, p/n 040-764) plus 1x DMSO Inhibitor Mix (Cell Biosciences, p/n 040-510) and
1x Aqueous Inhibitor Mix (Cell Biosciences, p/n 040-482).
Lysis details:
Wash cells with 10 mL of ice-cold PBS (Cellgro, cat# 21-031-CV), aspirate well. Add 400 µL ice-cold lysis buffer to 10-cm plate on
ice, swirl around to ensure good coverage, and incubate 10 minutes on ice. Scrape plate, pipet up-and-down to mix. Transfer lysate
to microfuge tube, lyse for an additional 30 minutes on ice. Clarify by centrifugation (14,000 x g, 15 minutes) in a cooled centrifuge.
Transfer supernatant to a fresh microfuge tube. Immediately aliquot supernatant (10-30 µL) on ice and snap freeze on dry ice.
Storage:
-80°C
Sample Preparation
Protein concentration:
0.08 mg/mL final in capillary by BCA assay
Sample diluent:
Sample Diluent (Cell Biosciences, p/n 040-649) plus 1x DMSO Inhibitor Mix
Ampholyte premix:
Premix 5-8 (nested) (Cell Biosciences, p/n 040-643)
pI Standards:
pI Standard Ladder 3 (Cell Biosciences, p/n 040-646)
Procedure:
Step 1) Dilute lysate with sample diluent to 0.16 mg/mL.
Step 2) In a separate tube, mix ampholyte premix and pI standards.
Step 3) Mix step 1 and step 2 at 1:1 to create final protein concentration.
Wash:
Wash Solution (Cell Biosciences, p/n 040-313)
Primary antibody:
Anti-PLCγ1 (Cell Signaling Technology, cat# 2822, 1:50), anti-phospho PLCγ1
(Abcam, cat# ab53125,1:50) in Antibody Diluent (Cell Biosciences, p/n 040-309)
Detection antibody:
Anti-Rabbit HRP (Cell Biosciences, p/n 040-656, 1:100) in Antibody Diluent
Anolyte:
Phosphoric Acid, 10 mM (Cell Biosciences, p/n 040-650)
Catholyte:
Sodium Hydroxide, 100 mM (Cell Biosciences, p/n 040-651)
Luminol/Peroxide:
Mixed 1:1 (Cell Biosciences, p/n 040-652 and p/n 040-653)
Assay Conditions
System:
NanoPro 1000 (formerly CB1000)
Focus conditions:
15000 μW, 40 minutes
Immobilization:
80 seconds
Wash 1:
2 x 150 seconds (default)
Primary antibody incubation:
120 minutes
Wash 2:
2 x 150 seconds (default)
Detection antibody incubation:
60 minutes
Wash 3:
2 x 150 seconds (default)
Chemiluminescence exposure:
60, 120, and 240 seconds