Result

HEK293 produced strong signal, In U87, HeLa and Jurkat cell lysates target bands were not detected. PKM2 antibody (STJ95142). (Figure: Lane1: HEK293; Lane 2: U87; Lane 3:HeLa; Lane 4: Jurkat).

Cell Line: HEK293,U87 cell lysate, HeLa

cell lysate, Jurkat

Method of validation: Western Blot

Primary Antibody: STJ95142 PKM2 antibody

Secondary Antibody Polyclonal Goat anti Rabbit- HRP

Conjugated (Dako)

Reference antibody: GAPDH (abcam)

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

E.g. 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 with 5% skimmed milk powder with 1:10000 dilution ratio, for 1.5 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).

Antibody Customer Review: STJ95142 Anti-PKM2 antibody Antibody Specificity:

Antibody Rating:

Gel electrophoresis information：12% polyacrylamide gel, constant voltage 185 V, 60 min.

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

No. Antigen Loading amount

Primary anti-body

Primary antibody dilution

ratio

Secondary

antibody dilu-tion ratio

Target band KD Visualization time

1 HEK293 cell

lysate 18µg(total

protein) STJ95142.

PKM2 anti-body

1:1000

1:5000 ~58KD 120S

2 U87 cell

lysate

18µg (total protein)

1:5000 ~58KD 120S

3 HeLa Cell lysate 18µg (total

protein)

STJ95142

PKM2 anti-body

1:1000 1:5000 ~58KD 120S

4 Jurkat Cell ly-

sate 18µg (total

protein)

STJ95142

PKM2 anti-body

1:1000 1:5000 ~58KD 120S

GAPDH

Marker 1 2 3 4