TXNIP Mouse Monoclonal Antibody [Clone ID: JY1]

Specifications

Product Data
Clone Name	JY1
Applications	IP, WB
Recommended Dilution	Western blotting: 10 µg/ml for chemiluminescence detection system. Immunoprecipitation: 2 µg/200 µl of cell extract from 5x10e6 cells­. Detailed procedure is provided in Protocols.
Reactivities	Human, Mouse
Host	Mouse
Isotype	IgG2b
Clonality	Monoclonal
Immunogen	Human recombinant VDUP-1/Txnip.
Specificity	This antibody reacts with VDUP-1 (50 kDa) by Western blotting.
Formulation	PBS, pH 7.2 containing 50% Glycerol without preservatives. State: Azide Free State: Liquid purified IgG fraction.
Concentration	lot specific
Purification	Protein-A Agarose Chromatography of hybridoma supernatant.
Conjugation	Unconjugated
Storage	Store the antibody (in aliquots) at -20°C. Avoid repeated freezing and thawing.
Stability	Shelf life: one year from despatch.
Gene Name	Homo sapiens thioredoxin interacting protein (TXNIP), transcript variant 1
Database Link	Entrez Gene 56338 MouseEntrez Gene 10628 Human UniProt ID Q9H3M7
Background	Vitamin D3 up-regulated protein 1 (VDUP-1), also called Thioredoxin (TRX)-interacting protein, is an endogenous inhibitor of TRX. Redox-dependent regulation of VDUP-1 by mitogenic factors through Reactive oxygen species (ROS) and the specific binding of VDUP-1 to the redox-sensitive cysteine-sulfide center of TRX modulate intracellular levels of ROS and the mitogenic activity of TRX.
Synonyms	Thioredoxin-interacting protein
Note	This product was originally produced by MBL International. Protocol: SDS-PAGE & Western Blotting: 1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) containing appropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the cold Lysis buffer to make an 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli’s sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at RT, or overnight at 4°C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1% skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The optimal antibody concentration will depend on the experimental conditions.) 8) Wash the membrane with PBS (5 minutes x 6 times). 9) Incubate the membrane with the 1/10,000 HRP-conjugated anti-Mouse IgG diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at RT. 10) Wash the membrane with PBS (5 minutes x 6 times). 11) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap. 12) Expose to an X-ray film in a dark room for 5 minutes. 13. Develop the film as usual. The conditions for exposure and development may vary. Positive Controls: Raji, K562, KG1, MRC5, IC2Tr, HEL, P19. Immunoprecipitation: 1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) containing appropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant to another tube. 3) Add primary antibody as suggested in the APPLICATIONS into 200 µL of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4°C. Add 20 µL of 50% protein A agarose beads resuspended in the cold Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4°C. 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 beads in 20 µL of Laemmli's sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Load 10 µL of the sample per lane in a 1mm thick SDS-polyacrylamide gel for electrophoresis. 6) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for specific transfer procedure. 7) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4°C. 8) Incubate the membrane with primary antibody diluted with PBS, pH7.2 containing 1% skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The optimal antibody concentration will depend on the experimental conditions.) 9) Wash the membrane with PBS (5 minutes x 6 times). 10) Incubate the membrane with the 1:5,000 HRP-conjugated anti-mouse IgG k light chain diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 11) Wash the membrane with PBS (5 minutes x 6 times). 12) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap. 13) Expose to X-ray film in a dark room for 5 minutes. Develop the film as usual. The conditions for exposure and development may vary. Positive Control: Raji.
Reference Data
Protein Families	Druggable Genome