M165-8 RFP antibody

RFP antibody  

RFP （3G5）-Agarose

 Application: WB ICC

Clonality:Monoclonal

BACKGROUND: Expression vector containing a tag sequence is commonly used to introduce and express a specific gene into a target cell. Red Fluorescent Protein （RFP） fusion protein expression system is preferably used in various laboratories, because it’s easy monitoring of fusion proteins. This specific antibody for RFP is useful tool for monitoring of the fusion protein expression.

 SOURCE: This antibody was purified from hybridoma （clone 3G5） supernatant using protein A agarose. This hybridoma was established by fusion of mouse myeloma cell P3U1 with C3H mouse lymphocyte immunized with RFP.

FORMULATION: 400 μg of anti-RFP monoclonal antibody covalently coupled to 200 μL of agarose gel and provided as a 50% gel slurry suspended in PBS containing preservative （0.09% sodium azide） for a total volume of 400 μL.

Azide may react with copper or lead in plumbing system to form explosive metal azides. Therefore, always flush plenty of water when disposing materials containing azide into drain.

*STORAGE: This antibody solution is stable for one year from the date of purchase when stored at 4oC.

REACTIVITY: This antibody reacts with RFP fusion proteins on Immunoprecipitation.

APPLICATION:

ImmunoprecipitationImmunoprecipitation; 20 μL

Detailed procedure is provided in the following PROTOCOL.

INTENDED USE:

For Research Use Only. Not for use in diagnostic procedures.

PROTOCOL:

Immunoprecipitation

1） Wash the transfectant cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer （50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40） containing appropriate protease inhibitors. Incubate it at 4oC with rotatin g f or 15 minutes, then sonicate briefly （up to 10 seconds）. Centrifuge the tube at 12,000 x g for

2）10 minutes at 4C and transfer the supernatant to another tube.

 3） Add primary antibody as suggest in the APPL in to 200 μL of cell extract. Mix well and incubate with gentle agitation for 60-120 minutes at 4oC.

4）Wash the beads 3-5 times with the cold Lysis buffer （centrifuge the tube at 2,500 x g for 10 seconds）.

5）Resuspend the agarose in 20 μL of Laemmli’s sample buffer, boil for 3-5 minutes, and centrifuge for

6）Load 10 μL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 7） Blot the protein to a polyvinylidene difluoride （PVDF） membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system （Transfer B 20% MeOH）. See the manufacture's manual for precise transfer procedure. 8） To reduce nonspecific binding, soaskimmed milk （in PBS, pH 7.2） for 1 hour at room temperature, or overnight at 4o

9） Wash the membrane with PBS-T [0.05% Tween-20 in PBS] （5 minutes x 3 times）.

10） Incubate the membrane with 1 μg/mL of anti-RFP monoclonal antibody （MBL; code no. M155-3） diluted with PBS, pH 7.2 contaiat room temperature. （The concentration of antibody will depend on condition.）

11）Wash the membrane with PBS-T [0.05% Tween-20 in PBS] （5 minutes x 3 times）. 12） Incubate the membrane with the 1:10,000 HRP-conjugated anti-mousdiluted with 1% skimmed milk （in PBS, pH 7.2） for hour at room temperature. 13） Wash the membrane with PBS-T （5 minutes x 3 times）. 14） Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 15） Expose to an X-ray filmdevelopment may vary.