RPPA Customer Review for Anti-Phospho-ErbB-3 (Y1197) Polyclonal Antibody (STJ90642)

HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: Phospho-ErbB-3 (Y1197) Polyclonal Antibody (STJ90642)

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

Reverse Phase Protein Array (RPPA)RPPA is a useful method for quantifying protein levels in cell or tissue lysate. The video belowDemonstrates how to perform a typical RPPA experiment. For more detailed information on

how RPPA was used to validate our antibody, see the following slides.

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).

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

What was the protocol?

No. Antigen Loading amount

Primary antibody

Primary antibody dilution

ratio

Secondaryantibody 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 Huh75 LX-76 MCF77 SEM8 U2OS

What were the results?

Figure: Alpha-tubulin normalised relative fluorescent intensity

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.

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