Result

“Looked promising initially but results don’t tally up with an antibody that is used and trusted in group (see bottom blot). Samples used overexpress Runx1 but very little difference between the Puro and Runx1 were seen with the St Johns Ab. Expected results are shown using the same blot with our usual antibody.” - J. Hay, University of Glasgow

Cell Line: GP86 Puro cells and Runx1P1 overexpressing cells (murine)

Method of validation: Western Blot

Primary Antibody: STJ96177 UCH-L1 Antibody

Dilution ratio: 1:1000

Positive Control: 3s+ a Runx1 expressing mu-

rine line

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 for 5 minutes and put on a heatblock at 95C̊.

Gel electrophoresis: 10% polyacrylamide gel, constant voltage of 160V for 60 minutes.

Transfer: 0.45 µm NC membrane, 1 piece of gel (semi dry transfer), constant voltage 20V 60 minutes.

Blocking: 1X PBST with 5% skimmed milk powder was used and left for 2 hours.

Primary antibody probing: Primary antibody was added to the solution of 1X PBST with 5% skimmed milk powder, 4C̊ for overnight.

Membrane wash: Membrane was washed 3 times with 1X PBST.

Secondary antibody probing: Secondary antibody was added to the solution OF 1X PBST with 5% skimmed milk powder with 1:3000 dilution ratio, for 1 hour.

Membrane wash: Membrane was washed 3 times with 1X PBST.

Visualization: ECL visualization for 5 minutes (the visualization time was determined by actual result. It was ensured that the visualization time was identical for the same antigen).

Antibody Customer Review: STJ25422 Anti-RUNX1 antibody Antibody Specificity:

Antibody Rating:

Gel electrophoresis information: 10% polyacrylamide gel, constant voltage of 160V for 60 minutes.

Transfer information: 0.45 µm NC membrane, 1 piece of gel (semi dry transfer), constant voltage 20V 60 minutes.

No. Antigen Loading amount

Primary anti-body

Primary antibody

dilution ratio

Secondary antibody dilu-

tion ratio Target band KD Visualization time

1 Puro (GP86) 50µg Anti-RUNX1

antibody (STJ25422

1:2000

1:20000 ~48KD 20s

2 Runx1 (GP86)

50µg Anti-RUNX1

antibody (STJ25422

1:2000 1:20000

~48KD

20s