HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: Anti-Caspase-3 Polyclonal Antibody (STJ90025)

What was used?Primary antibody: STJ90025 anti-Caspase-3 antibody

Provider: St John's Laboratory Ltd.

Dilution ratio: 1:500

Application: Western Blot

Materials for validation: Lysates from Hela cell line

Click for product data sheet PDF

What Was The Protocol?Treatment of Materials When cells were cultured to 70%-80% plating density, lysis buffer with protease inhibitors

(Sigma) and equal volume of 2X loading buffer were added and heated at 100˚C for 10 minutes. Supernatant was collected after centrifugation at 12,000 rpm for 25 minutes at 4°C and used for 10% Bis-Tris plus gel for protein separation.

Gel Electrophoresis 10% polyacrylamide gel, constant voltage 160 V, 60 minutes.

Transfer 0.45 µm PVDF membrane, 1 piece of gel (wet transfer), constant current 80 Amp for 90 minutes.

Blocking 1X Odyssey blocking solution for 2 hours at RT

Primary Antibody Probing Primary antibody was added to the 1X Odyssey blocking solution with 1:500 dilution, and the membrane was incubated at 4˚C overnight with agitation.

Membrane Wash 1X TBST wash 3 times

Secondary Antibody Probing Secondary antibody was added to 1X Odyssey blocking solution with 1:1000 dilution ratio, for 1.5 hours at RT

Membrane Wash 1X TBST wash 3 times

Visualization Fluorescent signals were visualized using a Li-Cor Odyseey machine using 700nm and 800nm detecting channels

What was the protocol?10% polyacrylamide gel, constant voltage 160 V, 60 minutes.

0.45 µm PVDF membrane, 1 piece of gel (wet transfer), constant current 80 Amp for 90 minutes.

No. Antigen Loading amount

Primary antibody

Primary antibody

dilution ratio

Secondaryantibody

dilution ratioTarget band KD Visualization

time

1 Hela lysate 10µg (total protein)

STJ96023 anti-Caspase-3 Antibody

1:500 1:0000 ~35KD N/A

2 ~46KD N/A1:0000Actin-beta 1:1000

What were the results?

What did the customer think?

Antibody Specificity:

Antibody RPPA Rating:

Testing results were provided independently by Junyoung Huang at Newcastle University. They stated “HeLa cell lysates were used. Western blot signals were detected using an Odyssey (Li-Cor) machine). A very weak protein band which was assumed to be the cleaved caspase-3 protein was detected around 21KD using anti-caspase-3 (STJ90025) antibody at 1:500 dilution and a rabbit Alexa fluor 680 secondary antibody. We couldn’t detect any protein bands around 35KD position as it was suggested from the provided data sheet. ”

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