Result “In mouse striatal progenitors a target band was detected by STJ95200 PPAR-α Anbody. The signal aﬅer ECL detecon was very week, stronger diluon (1:500) of anbody might be needed .” - K. Palm, Protobios. Gel electrophoresis informaon：10% polyacrylamide gel, electric current 35 mA, 90 minutes. Transfer informaon：0.45 um PVDF membrane, 1 piece of gel (wet transfer), electric current 350 mA 95 minutes. Lane No. Angen Loading amount Primary anbody Primary anbody diluon rao Secondary anbody diluon rao Target band KD Visualiza on me 1 Striatal progenitors 10 ug STJ95200 An- PPAR-α anbody 1:1000 1:10000 ~50KD 2 minutes 2 Sriatal progenitors treated with 20 nM troglitazone 10 ug STJ95200 An- PPAR-α anbody 1:1000 1:10000 ~50KD 2 minutes Cell Line: Mouse striatal progenitors Method of validaon: Western Blot Primary Anbody: STJ95200 An-PPAR-α anbody Secondary Anbody HRP goat an-rabbit Reference anbody: LaminB1 (Abcam) Diluon rao: 1:1000 Protocol Treatment of materials: Cells were cultured to 70%-80% plang density, cytoplasmic fracon was extracted by hypotonic buffer (10 mM Hepes, 1,5 mM MgCl, 10 mM KCL, 0,5 mM DTT, ph 7,9) with protease inhibitors, 5x loading buffer was added, denaturaon was performed at 50 ˚ C for 10 minutes. Gel electrophoresis: 10% polyacrylamide gel, electric current 35 mA, 90 minutes. Transfer: 0.45 um PVDF membrane, 1 piece of gel (wet transfer), electric current 350 mA 95 minutes. Blocking: 1X TBS Tween (0,1%) with 5% skimmed milk powder for 1 hour. Primary anbody probing: primary anbody was added to the soluon of 1X TBS Tween (0,1%) with 5% skimmed milk powder, 4˚ C for overnight. Membrane wash: 1X TBS Tween (0.1%) wash for 3 mes. Secondary anbody probing: secondary anbody was added to the soluon of 1X TBS Tween (0,1%) with 5% skimmed milk powder with 1:10000 diluon rao, for 1 hour. Membrane wash: 1X TBS Tween (0.1%) wash for 3 mes. Visualizaon: ECL visualizaon for 5 minutes with SuperSignal West Pico ECL-kit (Thermo scienfic) Anbody Customer Review: STJ95200 An-PPAR-α anbody Anbody Specificity: Anbody Rang: Figure: line 1 control, line 2 treatment of 20 nM troglitazone

Western Blot Antibody Customer Review for Anti-PPAR-α Polyclonal Antibody (STJ95200)

Download PDF Report

Upload
st-johns-laboratory-ltd
View
59
Download
1

Embed Size (px)

Citation preview

Page 1: Western Blot Antibody Customer Review for Anti-PPAR-α Polyclonal Antibody (STJ95200)

Result

“In mouse striatal progenitors a target band was detected by STJ95200 PPAR-α Antibody. The signal after ECL detection was very week, stronger dilution (1:500) of antibody might be needed .” - K. Palm, Protobios.

Gel electrophoresis information：10% polyacrylamide gel, electric current 35 mA, 90 minutes.

Transfer information：0.45 um PVDF membrane, 1 piece of gel (wet transfer), electric current 350 mA 95 minutes.

Lane No. Antigen Loading amount

Primary antibody

dilution ratio

Secondary

antibody dilution ratio

Target band KD Visualizati

on time

1 Striatal

progenitors 10 ug

STJ95200 Anti-PPAR-α

antibody 1:1000 1:10000 ~50KD 2 minutes

Sriatal progenitors

treated with 20 nM troglitazone

10 ug STJ95200 Anti-

PPAR-α antibody

1:1000 1:10000 ~50KD 2 minutes

Cell Line: Mouse striatal progenitors

Method of validation: Western Blot

Primary Antibody: STJ95200 Anti-PPAR-α

antibody

Secondary Antibody HRP goat anti-rabbit

Reference antibody: LaminB1 (Abcam)

Dilution ratio: 1:1000

Protocol

Treatment of materials:

Cells were cultured to 70%-80% plating density, cytoplasmic fraction was extracted by hypotonic buffer (10 mM Hepes, 1,5 mM MgCl, 10 mM KCL, 0,5 mM DTT, ph 7,9) with protease inhibitors, 5x loading buffer was added, denaturation was performed at 50 C̊ for 10 minutes.

Gel electrophoresis: 10% polyacrylamide gel, electric current 35 mA, 90 minutes.

Transfer: 0.45 um PVDF membrane, 1 piece of gel (wet transfer), electric current 350 mA 95 minutes.

Blocking: 1X TBS Tween (0,1%) with 5% skimmed milk powder for 1 hour.

Primary antibody probing: primary antibody was added to the solution of 1X TBS Tween (0,1%) with 5% skimmed milk powder, 4C̊ for overnight.

Membrane wash: 1X TBS Tween (0.1%) wash for 3 times.

Secondary antibody probing: secondary antibody was added to the solution of 1X TBS Tween (0,1%) with 5% skimmed milk powder with 1:10000 dilution ratio, for 1 hour.

Membrane wash: 1X TBS Tween (0.1%) wash for 3 times.

Visualization: ECL visualization for 5 minutes with SuperSignal West Pico ECL-kit (Thermo scientific)

Antibody Customer Review: STJ95200 Anti-PPAR-α antibody Antibody Specificity:

Antibody Rating:

Figure: line 1 control, line 2 treatment of 20 nM

troglitazone