HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: Anti-Actin β Polyclonal Antibody (STJ91464)

What was used?Primary antibody: STJ91464 Anti-Actin β Antibody

Provider: St John's Laboratory Ltd.

Dilution ratio: 1:500

Application: Reverse Phase Protein Array (RPPA)

Materials for validation: Cellular lysates of the human fibroblast cells with TGF-beta treatment over 0-180mins in triplicate (customer provided samples). Samples with alkaline phosphatase treatment from another cell line and parallel control (no AP treated)

Click for product data sheet PDF

What was the protocol?Treatment of Materials

1. Cells cultured to 80% plating density.2. RPPA lysis buffer (with protease inhibitor) applied and samples sonicated with Bioruptor at 15s on/off frequency for total of 15mins.3. Samples centrifuged at 12000RPM for 5mins. 4. Supernatants collected and protein quantification using Bradford assay (Sigma). 5. Control samples from another cell line were then split out for Alkaline Phosphatase treatment at 37 ˚C for 60 mins. 6. Original samples with and without Alkaline Phosphatase treatment adjusted to a ratio of 1:5 dilution 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. 7. Prepared samples were printed onto ZeptoChIP using GESIM Nanoplotter 2.1 in duplicate at 50% humidity at 16 ˚C chilling condition. 8. Printed chips were incubated with blocking buffer (containing BSA) in nebuliser statically to allow sufficient blocking for 1hour at room temperature. 9. Blocked chips were washed in deionised ultrapure water x 3 and spin-dried at 300 RPM. 10. Chips were loaded into the fluidic structure and washed x 3 to equilibrate the incubation chambers and allow efficient overnight incubation of primary antibodies at 4 ˚C. 11. Secondary antibody incubation was carried out for 2.5 hours at 4 ˚C. 12. Final readout through ZeptoREADER at 3 exposure times (10s, 5s, 1s).

Sample Preparation 1:5 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 hours at 4˚C.

Visualisation Assay Buffer wash for 3 times and visualization carried out using ZeptoREADER

What was the protocol?

RPPA platform: Zeptosens

No. Antigen Loading amount

Primary antibody

Primary antibody dilution

ratio

Secondaryantibody

dilution ratioTarget band KD Visualization

time

1 Human fibroblast cells

500pl/per spot

STJ91464 Actin beta 1:500 1:500

N/A(RPPA does not tell

protein size)

10S,5S,1S

What were the results?

Figure: Relative fluorescent intensity of actin beta on sample sets

What did the customer think?

Testing results were provided independently by Dr N. Wang of Newcastle University.

Raw images were analysed using ZeptoVIEW software for automated image examination and evaluation. Image quality was controlled by the follow factors: saturated pixel, spot intensity, eccentricity, donu effect, signal-to-noise ratio, discovered diameter. Final scores for individual samples (a sum of 8 to represent 1 sample) were categorised into Good, Undet, Bad. Data were exported into Excel format (RFI and RNFI) and further analysed using R statistic software for further antibody evaluation. No rating supported for total antibody but a linear fit of over 0.7 between RPPA and WB is regarded as RPPA usable antibody.

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