Result

“There are two bands around 39 and 43 KDs were detectable. Presumable, the lower band is the Mcl-1 which is close to the given size. We have used 1:500 dilutions, the signal wasn’t very strong, but two bands around 39 and 43 KDa can be detected and the background is reasonable. “ - Jun-Yong Huang, Newcastle University

Gel electrophoresis information：10% polyacrylamide gel, constant voltage 160 V, 60 min.

Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 80Amp.

Lane No. Antigen Loading amount

Primary anti-body

Primary anti-body dilution

ratio

Secondary

antibody dilution ratio

Target band KD Visualiza-tion time

1 Hela lysate 10 ug STJ94044 1:500 1:1000 ~37KD /

Cell Line: Lysates from Hela cell line

Method of validation: Western Blot

Primary Antibody: STJ94044 Anti-Mcl-1

Secondary Antibody Alexa fluor 680 rabbit IgG

(H+L) antibody

Dilution ratio: 1:500

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 heating at 100C̊ for 10 min. Supernatant was collected after centrifuged at 12,000 rpm for 25 min at 4oC and used for 10% Bis-Tris plus gel for protein separation.

E.g. Gel electrophoresis: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant current 80 Amp 90 min.

Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 90 min.

Blocking: 1X Odyssey blocking solution for 2 hr 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 4C̊ for overnight with agitation.

Membrane wash: 1X TBST wash for 3 times.

Secondary antibody probing: secondary antibody was added to 1X Odyssey blocking solution with 1:500 dilution with 1:1000 dilution ratio, for 1.5 hr at RT.

Membrane wash: 1X TBST wash for 3 times.

Visualization: : Fluorescent signals were visualization using a Li-Cor Odyseey machine using 700nm and 800nm de-tecting channels.

Antibody Customer Review: STJ94044 Anti-Mcl-1 Antibody Antibody Specificity:

Antibody Rating: 1319

7

10

4

5

2

3

2

(KD)

Mcl-1