RA18003
3Pages

APP Datasheet Host: Rabbit Species Reactivity: Rat, Mouse, Human, Monkey Format: Liquid in 10mM sodium HEPES (pH7.5), 150mM NaCl, 100ug bSa and 50% glycerol Product Type: Affinity purified antibody Immunogen Sequence: Peptide corresponding to residues surrounding Thr668 of human APP695. Antibodies are purified by protein A and peptide affinity chromatography. Applications: Western blotting 1:1000 Immunohistochemistry (paraffin) 1:50 Dilutions listed only as a recommendation. Optimal dilution should be determined by investigator. Storage: Store at -20°C. Do not aliquot. Application Notes APP Antibody detects endogenous levels of several isoforms of both mature and immature amyloid p (A4) precursor protein, including APP695, APP770 and APP751. Western Blot Protocol Sample Preparation 1. Treat cells by adding fresh media containing regulator for desired time. 2. Aspirate media from cultures; wash cells with 1X PBS; aspirate. 3. Lyse cells by adding 1X SDS sample buffer (100 pl per well of 6-well plate or 500 pl per plate of 10 cm plate). Immediately scrape cells from plate and transfer the extract to a microcentrifuge tube. Keep on ice. 4. Sonicate for 10-15 seconds to shear DNA and reduce sample viscosity. 5. Heat a 20 pl sample to 95-100°C for 5 minutes; cool on ice. 7. Load 20 pl onto SDS-PAGE gel (10 cm x 10 cm). 8. Electrotransfer to nitrocellulose (or PVDF) membrane. Membrane Blocking and Antibody Incubations Note: Volumes for 10 cm x 10 cm (100 cm2) membrane; for different sized membranes, adjust vol. accordingly. 1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 minutes at room temperature. 2. Incubate membrane in 25 ml of blocking buffer for 1 hour at room temperature. 3. Wash three times for 5 minutes each with 15 ml of TBS/T. 4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C. 5. Wash three times for 5 minutes each with 15 ml of TBS/T. 6. Incubate membrane with HRP-conjugated secondary antibody (1:2000) in 10 ml of blocking buffer with gentle agitation for 1 hour at room temperature. 7. Wash three times for 5 minutes each with 15 ml of TBS/T. 8. Process membranes using enhanced chemiluminescence. FOR RESEARCH USE ONLY Data and Protocol Provided Courtesy of Cell Signaling Technology, Inc. Neuromics reagents are for in vitro and certain non-human in vivo experimental use only and not intended for use in any human clinical investigation, DIAGNOSIS, PROGNOSIS, OR TREATMENT. THE ABOVE ANALYSES ARE MERELY TYPICAL GUIDES. THEY ARE NOT TO BE CONSTRUED AS BEING SPECIFICATIONS. ALL OF THE ABOVE INFORMATION IS, TO THE BEST OF OUR KNOWLEDGE, TRUE AND ACCURATE. HOWEVER, SINCE THE CONDITIONS OF USE ARE BEYOND OUR CONTROL, ALL RECOMMENDATIONS OR SUGGESTIONS ARE MADE WITHOUT GUARANTEE, EXPRESS OR IMPLIED, ON OUR PART. WE DISCLAIM ALL LIABILITY IN CONNECTION WITH THE USE OF THE INFORMATION CONTAINED HEREIN OR OTHERWISE, AND ALL SUCH RISKS ARE ASSUMED BY THE USER. WE FURTHER EXPRESSLY DISCLAIM ALL WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. www.neuromics.com Neuromics Antibodies • 5325 West 74th Street, Suite 8 • Edina, MN 55439 phone 866-350-1500 • fax 612-677-3976 • e-mail pshuster@neuromics1.com 6/04v1

Data Sheet Solutions and Reagents for Western Blot Note: Prepare solutions with Milli-Q or equivalently purified water. 1X Phosphate Buffered Saline (PBS) 1X SDS Sample Buffer: 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5) 10X Tris Buffered Saline (TBS): To prepare 1 liter of 10X TBS: 24.2 g Tris base, 80 g NaCl; adjust pH to 7.6 with HCl (use at 1X). Nonfat Dry Milk (weight to volume [w/v]) Blocking Buffer: 1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry...

Data Sheet Deparaffinize/hydrate sections: a. Incubate sections in three washes of xylene for 5 minutes each. b. Incubate sections in two washes of 100% ethanol for 10 minutes each. c. Incubate sections in two washes of 95% ethanol for 10 minutes each. Wash sections twice in dH2O for 5 minutes each. Wash sections in PBS for 5 minutes. For antigen unmasking, heat sections in microwave in 10 mM sodium citrate buffer (pH 6.0) for 1 minute at full power followed by 9 minutes at medium power. (Keep slides fully immersed in buffer and maintain temperature at or just below boiling. Exact microwave...