western blot antibody customer review for anti-cleaved-caspase-3 p17 (d175) polyclonal antibody...

HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: Anti-Cleaved-Caspase-3 p17 (D175) Polyclonal Antibody (STJ90026)

What was used?Primary antibody: STJ90026 Anti-Cleaved-Caspase-3 p17 (D175) 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)

STJ90026 Anti-Cleaved-

Caspase-3 p17 (D175)

1:500 1:0000 ~17KD N/A

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

What were the results?(KD)

46

32

57

76

100

134

2217

25

Actin

Caspase-3P17 (D175)

***

*

STJ90026

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). Assumed cleaved caspase-3 protein band was detected at the position of 20KD (in red) and is slightly higher than the suggested 17KD from provided data sheet by anti-caspase-3 p17 (D175, STJ90026) antibody with 1:500 dilution) and a rabbit Alexa fluor 680 secondary antibody. The antibody also detected several protein bands (in red) above 32KD, which have not been mentioned from the provided data sheet. An actin band is also illustrated in green at 46KD using the actin antibody from Abcam (ab6276) and the mouse Dylight 800 conjugated secondary antibody.”

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