Receptor tyrosine-protein kinase erB-3 (also known as human epidermal growth factor 3) is a protein encoded by the ERBB3 gene in humans. It is a membrane bound protein that is a part of the receptor tyrosine kinases family. Its role is to act as a receptor for neuregulins whilst on the cell surface. The Phospho-ErbB-3 (Y1197) polyclonal antibody detects endogenous levels of ErbB-3 protein when phosphorylated at Y1197. Anti-Phospho-ErbB-3 (Y1197) antibody - http://www.stjohnslabs.com/phospho-erbb-3-y1197-antibody?filter_name=STJ90642 Join Our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

1. HOW DID OUR
ANTIBODY PERFORM?
ANTIBODY CUSTOMER REVIEW: Phospho-ErbB-3 (Y1197) Polyclonal Antibody
(STJ90642)

2. What was used?
Primary antibody: Phospho-ErbB-3 antibody (STJ90642)
Provider: St John's Laboratory
Dilution ratio: 1:500
Application: Reverse Phase Protein Array (RPPA)
Materials for validation: Cellular lysates from h293T, Huh7,
LX-7, MCF7, SEM and U2OS cell lines
Click for product data sheet PDF

3. Reverse Phase Protein Array (RPPA)
RPPA is a useful method for quantifying protein levels in cell or tissue lysate. The video below
Demonstrates how to perform a typical RPPA experiment. For more detailed information on
how RPPA was used to validate our antibody, see the following slides.

4. What was the protocol?
Treatment of Materials When cells were cultured to 80% plating density, RPPA lysis buffer (with protease
inhibitor) was applied and samples were then collected and sonicated with
Bioruptor at 15s on/off frequency for a total of 15mins. Samples were then
centrifuged at 12000RPM for 5mins. Supernatants were collected and total
protein quantification was carried out using Bradford assay (Sigma). Samples were
then split out for Alkaline Phosphatase treatment at 37 C for 50 mins. Original
samples without Alkaline Phosphatase treatment and Alkaline Phosphatase
treated samples were all adjusted to 0.2mg/ml and 0.1mg/ml concentrations
respectively at a ratio of 1:10 between lysis buffer and spotting buffer and serially
diluted to 75%, 50%, 25% of original concentration on 384 well plates using
Biomek 3000 liquid handler. Prepared samples were then printed onto ZeptoChIP
using GESIM Nanoplotter 2.1 in duplicate at 50% humidity at 16 ˚C chilling
condition. Printed chips were then incubated with blocking buffer (BSA
containing) in nebuliser statically to allow sufficient blocking for 1hour at room
temperature. Blocked chips were then washed in deionised ultrapure water three
times and spin-dried at 300 RPM. Chips were then loaded into the fluidic structure
and washed three times to equilibrate the incubation chambers to allow efficient
overnight incubation of primary antibodies at 4 ˚C. Secondary antibody incubation
was carried out for 2.5 hours at 4 ˚C. Final readout was achieved through
ZeptoREADER at three exposure times (10s, 5s, 1s).

5. Sample Preparation 1:10 dilution of lysates in RPPA Spotting Buffer. Final protein
concentrations were equalized to 0.2mg/ml and 0.1mg/ml.
Serial Dilution All samples in 0.2mg/ml form were further diluted to 75%, 50%, 25% in
384-well plates.
Printing Samples were printed in duplicate using a nano-printer and printing
volumes were setup at 500 picolitre at 50% humidity at 16 ˚C.
Blocking Blocking was performed in a nebulizer with BSA containing Blocking
Buffer.
Chip Wash & Loading Chips were loaded into a fluidic structure and pre-washed three times in
Assay Buffer before primary antibody incubation.
Antibody Application Primary antibodies were diluted at 1:500in Assay Buffer and incubated 4˚C
for overnight, followed by an Assay Buffer wash for 3 times. Secondary
antibody was diluted 1:500 in Assay Buffer added to chips and incubated
for 2 hr at 4˚C.
Visualisation Assay Buffer wash for 3 times and visualization carried out using
ZeptoREADER

6. What was the protocol?
No. Antigen
Loading
amount
Primary
antibody
Primary
antibody
dilution
ratio
Secondary
antibody
dilution
ratio
Target band
KD
Visualization
time
1 h293T
500pl/per
spot
STJ90642
HER3
(phospho_
Y1197)
1:500 1:500
N/A
(RPPA does
not tell
protein size)
10S,
5S,
1S
3 Huh7
5 LX-7
6 MCF7
7 SEM
8 U2OS

7. What were the results?
Figure: Alpha-tubulin normalised relative fluorescent intensity

8. What did the customer think?
Antibody Specificity:
Antibody RPPA Rating:
Testing results were provided independently by Dr Nan Wang
at Newcastle University.
24/30
Rating based upon internal RPPA antibody validation protocol and RPPA scoring algorithm taking into account
weighted sum of scores including spots quality, signal-to-noise ratio, normalised fold-dilution fitting, normalised
fold-change with alkaline phosphatase treatment, reference spots effect and graininess, sparks and flares of
images. A Grade antibody (RPPA approved) scored between 20-30. B Grade antibody (RPPA usable) scored
between15-19. C Grade antibody (RPPA void) scored between 0-14.

9. JOIN THE ANTIBODY VALIDATION PROJECT

10. 1. Order any STJ9 catalogued product using this online form.
2. Test it in your laboratory using your preferred method.
3. Return a fully completed review form to receive rewards.
…in 3 simple steps…

11. @StJohnsLabs
http://www.slideshare.net/stjohnslabs