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RIP2 or RIPK2 is a kinase that plays a role in the modulation of the innate and and adaptive immune responses. Upon bacterial infection, RIP2 is activated, which in turn activates a signalling cascade that results in the activation of the transcription factor NF-Kappa-B, which interacts with DNA directly in the nucleus of the cell, to activate the expression of hundreds of genes required for the cell’s immune response, protection against apoptosis and growth control.
1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: Phospho-RIP2 (S176) Polyclonal Antibody (STJ90957)
2. 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 lysates Click for product data sheet PDF
3. 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
4. What was the protocol? Gel electrophoresis information, Gel 1 and £º7.5 % 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 Secondary antibody dilution ratio Target band kDa Visualization time 1 HCT116, No stimulation 10 ug STJ90957 Lanes 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
5. What were the results? (St John¡¯s Laboratory)
6. What did the customer think? Antibody Specificity: Antibody Rating:
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