b'Specialty detectionTable 27. Primary antibodies recognized by Clean-Blot IP Detection Reagent.reagents Species Isotype*Bovine IgG2Clean-Blot IP Detection Reagent Goat IgG2Thermo Scientific Clean-Blot IP Detection ReagentHuman IgG1, IgG2, IgG4is an HRP conjugate that is optimized for post- Mouse IgG2a, IgG2b, IgG3immunoprecipitation western blot detection of primaryRat IgG2cantibodies without interference from denaturedSheep IgG2immunoprecipitation (IP) antibody fragments. * Clean-Blot IP Detection Reagent recognizes polyclonal and monoclonal antibodies. To determine specific antibody compatibility, perform a dot-blot analysis.The Clean-Blot IP Reagent allows trouble-free western blot detection of target proteins following IP assays. It works by ) )specifically binding to functional primary antibodies (wholeRP RPH Hk1 RP k1 gent ( gent (IgG) without also binding to fragments of the IP antibodies, RP d d a ause H nti-C ouse H nti-C -Blot t on Re Blot tion Rewhich usually accompany the immunoprecipitated protein through electrophoresis and membrane transfer.i-mo use a nti-m ouse a lean etec i lean- etecThe Clean-Blot IP Reagent and Kit eliminate detectionAnt Mo + a M + C D C Dinterference from both heavy-chain (approximately 50 kDa) and light-chain (25 kDa) IgG fragments of antibodies usedHeavy chain (55 kDa)for the initial immunoprecipitation assay.Cdk1 (34 kDa)Features: Universalbind and detect most IgG isotypes andLight chain (22 kDa)subclasses of primary antibodies that are commonly used for western blotting (Table 27) Figure 56. Easily distinguish your target protein on a western blot with Clean-Blot Detection Reagent (HRP). Mouse liver extract (50 g)Compatibleeffective with IP assays performed usingtotal protein was separated on a Bio-Rad Criterion Gel, transferred to protein A, protein G, or anti-IgG agarose beads and anyPVDF membrane, and blocked with 5% milk in TBST. The membrane was blocking buffer probed with mouse monoclonal anti-Cdk1 (LabVision, 0.2 g/mL) and goat anti-mouse HRP (0.16 g/mL) or Clean-Blot Detection Reagent (HRP) (0.2 g/mL). SuperSignal West Pico substrate was used for detection ofCost-effectiveeliminates the cost and extra workCdk1 protein.associated with covalently immobilizing IP antibodies as a means of overcoming western blot interference A B C DClean-Blot Clean-Blot FlexibleHRP reagent for detection withGoat anti-rabbitDetectionGoat anti-rabbitDetection chemiluminescent, fluorescent, or colorimetric substrates HRP Reagent HRP Reagent Easy to useno need to change the western blotting protocol; simply replace conventional secondary HRPNFBconjugate with the Clean-Blot IP Detection Reagent (Figure 56)Bax Unobstructed detectionclear western blot results Lysate Lysate Lysate Lysatefor immunoprecipitation assays without significantWash Elute Wash Elute Wash Elute Wash Eluteinterference from denatured IgG bands (Figure 57)Figure 57. Reveal your target protein with Clean-Blot Detection Reagent (HRP). To demonstrate unmasking of the target protein, we performed IP and western blot experiments. NF-B and Bax were immunoprecipitated from A549 lysate using protein A/G agarose resin and (A and B) rabbit antiNF-B and (C and D) rabbit anti-Bax. (A and C) Detection was performed with goat anti-rabbit HRP, which masked the target. (B and D) Detection was performed with the Clean-Blot Detection Reagent (HRP), revealing the target protein.77'