HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: Phospho-RIP2 (S176) Polyclonal Antibody (STJ90957)

What was used?Primary antibody:

Phospho-RIP2 (S176) Polyclonal antibody (STJ90957)

Provider: St John's Laboratory

Dilution ratio: 1:1000

Application: Western Blot

Materials for validation: Mouse liver/colon lysatesClick for product data sheet PDF

What was the protocol?Treatment of Materials Cells were lysed in 1.5 mls of 1X standard RIPA lysis buffer with protease

inhibitors (PMSF and aprotinin) added. After lysis, samples were spun down and 4X sample buffer added. About 35 ul of the lysed cells was separated by SDS-PAGE at 10% and then transferred and blotted

Gel Electrophoresis 10% polyacrylamide gel, constant voltage 120V, 80 min. 50 ug per lane.

Transfer Nitrocellulose membrane

Blocking 1X TBST with 10% skimmed milk powder for 45 minutes.

Primary Antibody Application 1:1000 primary antibody was added to the solution of 1X TBST with 2.5% skimmed milk powder, 4˚C for overnight.

Secondary Antibody Application Secondary antibody (anti-rabbit HRP) was added to the solution of 1X TBST with 2.5% skimmed milk powder with 1:7500 dilution ratio, for 1.5 hours.

Visualisation ECL kit

What was the protocol?Gel electrophoresis information, Gel 1 and ： 7.5 % polyacrylamide gel, constant voltage 460 mA, 120 min.Transfer information: PVDF membrane, 1 piece of gel (wet transfer).

Gel No. Antigen Loading amount Primary antibody Primary antibody dilution ratio

Secondaryantibody dilution

ratioTarget band kDa Visualization time

1 HCT116, No stimulation 10 ugSTJ90957Lanes 1-5;

Anti-RIPK2 pS176 (Cell signaling)

Lanes 6-10

As indicated

1:7500 See below See above

2 HCT116, MDP 10 ug

3 IBC cells 10 ug 4 SUM149 cells 10 ug 5 BT549 Cells 10 ug

6 HCT116, No stimulation 10 ug

7 HCT116, MDP 10 ug

8 IBC cells 10 ug 9 SUM149 cells 10 ug

10 BT549 Cells 10 ug

What were the results?

(St John’s Laboratory)

What did the customer think?

Antibody Specificity:

Antibody Rating:

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