Result

“Excellent band of ubiquitin at 12 kd. Also showing other bands at different molecular weight. It may be non specific or its showing different ubiquitinated proteins. Need further Validations. ” G. P. Maiti, External Laboratory.

Gel electrophoresis information：7.5% polyacrylamide gel, constant voltage 160 V, 60 min.

Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 20V, overnight .

Lane No. Antigen Loading amount

Primary anti-body

Primary anti-body dilution

ratio

Secondary

antibody dilution ratio

Target band KD Visualiza-tion time

7 Whole cell ly-sate

20 ug STJ96863 Anti-Ub Antibody

1:1000 1:5000 ~145KD 1Min

Cell Line: Lymphoma cell lines

Method of validation: Western Blot

Primary Antibody: STJ96863 Anti-Ub Antibody

Secondary Antibody Sheep Anti Rabit

Dilution ratio: 1:1000

Protocol

Treatment of materials: None;

E.g. Gel electrophoresis: 7.5% polyacrylamide gel, constant voltage 20 V, overnight;

Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 20 V overnightl

Blocking: 1X TBST with 5% BSA for 2 hr.

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

Membrane wash: 1X TBST wash for 3 times.

Secondary antibody probing: secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with 1:5000 dilution ratio, for 1hr at room temperature.

Membrane wash: 1X TBST wash for 3 times.

Visualization: ECL visualization for 1 minute.

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Fig.1 - Figure showing the expression of Ub in different Lymphoma cell lines