Result

“HEK293 cell lysate produced strong signal, In U87,HeLa and Jurkat cell lysates target bands were not detected. PDHA-1 antibody (STJ950006). Dilution was 1:1000 and there were no visibility of background signals. “ - H. Kramer University of Oxford

Cell Line: HEK293 cell lysate, U87 cell lysate,

Method of validation: Western Blot

Primary Antibody: STJ95006 PDHA1 antibody

Secondary Antibody Goat Anti-Rabbit HRP Conjugated

Reference antibody: GAPDH

Dilution ratio: 1:1000

Protocol

Treatment of materials:

Cell pellets were lysed in RIPA lysis buffer with protease inhibitor cocktail (Roche) and equal volume of 2X loading buffer (Sigma) were heated at 95C̊ for 10 min in a heat block

Gel electrophoresis: 12% polyacrylamide gel, constant voltage 150 V, 60 min.

Transfer: 0.45 µm NC membrane, 1 piece of gel (wet transfer), constant voltage 40 V 120 min.

Blocking: 1X TBST with 5% skimmed milk powder over night.

Primary antibody probing: primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, incubation on the shaker, room temperature for 1hr.

Membrane wash: 1X TBST wash for 3 times with each time 5minutes incubation on the shaker.

Secondary antibody probing: secondary antibody was added to the solution of 1X TBST+ 5% skimmed milk powder with 1:5000 dilution ratio with mouse mAb for blue ladder (dilution 1:5000) for 1 hr.

Membrane wash: 1X TBST wash for 3 times with each time 5minutes incubation on the shaker and final wash with deionized wa-ter. Visualization: ECL visualization for 5 min (the visualization time was determined by actual result. It was ensured that the visualiza-tion time was identical for the same antigen). Visualization: ECL visualization for 5 min (the visualization time was determined by actual result. It was ensured that the visualization time was identical for the same antigen).

Antibody Customer Review: STJ95006 Anti-PDHA1 Antibody

Antibody Specificity:

Antibody Rating:

No. Antigen Loading amount Primary anti-

body

Primary antibody

dilution ratio

Secondary

antibody dilution ratio

Target band KD Visualization time

1 HEK293 cell

lysate 18µg(total pro-

tein) STJ95006 anti PDHA1 anti-

body 1:1000

1:5000 ~43KD 60S

2 U87 cell

lysate

18µg (total protein)

1:5000 ~43KD 60S

3 HeLa cell lysate 18 µg (total

protein)

STJ95006 anti-PDHA1 anti-

body 1:1000 1:5000 ~43KD 60S

4 Jurkat cell lysate 18 µg (total

protein)

STJ95006 anti-PDHA1 anti-

body 1:1000 1:5000 ~43KD 60S

Gel electrophoresis information：12% polyacrylamide gel, constant voltage 150 V, 60 min.

Transfer information：0.45 µm NC membrane (Bio Rad), 1 piece of gel (wet transfer), constant voltage 40 V 120 min.

Figure: Lane1: HEK293; Lane 2: U87; Lane 3: HeLa; Lane 4: (Jurkat).

Marker 1 2 3 4

GAPDH