“We believe that full-length cDNAs isolated from cDNA libraries are the most reliable source of authentic coding regions for our critical applications." said Drew Murphy, Vice President of Target Discovery for Regeneron. "The OriGene True Clone collection has proven to be an excellent source of full-length human cDNAs for use in Regeneron's protein expression and target validation platforms."
Antibody Use Protocol
Cell Lysing Protocol:
Remove the culture media by aspiration.
Wash the cells in the dish once with ice-cold PBS and aspirate off PBS.
Add ice-cold NP40 Cell Lysis Buffer*. We recommend adding 0.8 to 1 ml NP40 Cell Lysis buffer for 10 cm cell culture dish, when cells are over 70% confluent.
For adherent cells, scrap cells using cell scraper (Fisherbrand Disposable Cell Lifter, Fisher Scientific, 08-773-1) under ice. For suspension cells, pellet the cells, then resuspend in lysis buffer under ice.
Transfer the cell lysis solution into eppendorf tubes after pipetting the cell pellets 15 times.
Sonicate the cell lysates under ice for 10 sec.
Centrifuge the lysate at 12,000g in a pre-cooled centrifuge for 15 minutes.
Immediately transfer the supernatant to a fresh centrifuge tube and discard the pellet.
Determine the protein concentration by any commercially available reagent kit. We recommend using Ouant-iT Protein Assay Kit (Invitrogen, Q33210).
At this step, the sample can be divided into aliquots and stored at –80o C for long-term.
*NP40 Cell Lysis Buffer:
50 mM Tris-HCl pH 7.5 1% P40 (NP40) 250 mM NaCl 5 mM EDTA pH 8.0 50 mM NaF
Add fresh: 1 mM PMSF, 1 mM Na3VO4, and 1 X Protease Inhibitor Cocktail (P2714, Sigma).
Sample Preparation, Electrophoresis and Protein Transferring:
Add 5 µg of the above prepared cell lysate into sample buffer in a volume of total 20 µL (for a mini-gel, up to 15 ug of protein can be loaded per lane). We recommend using Invitrogen Nupage LDS Sample Buffer (NP0007). If reducing condition is needed, add 50 mM DTT into the sample tube.
Heat the sample to 70o C for 10 min. Prepare a pre-stained protein standard (SeeBlue Plus 2 Prestained Standard, Invitrogen, LC5925) as well.
Run the samples on a pre-cast SDS polyacrylamide gel at 170V (constant voltage) for 40 to 60 minutes until the dye reaches the bottom the gel. We recommend using 4-12% Bis-Tris gel (Invitrogen, NP0323BOX) for proteins with molecular weights smaller than 200 kD.
Remove the gel and soak in protein transfer buffer* for 15 minutes. Cut the nitrocellulose membrane (ISC BioExpress, F-3139-3) to the similar size of the transfer area of the gel. Assemble the electroblotting cassette and place the electrodes in the blotting unit, according to the manufacture’s instructions.
Transfer in Tris-Glycine transfer buffer at 100 V for 1 hour at constant current (not to exceed 0.4 A).
Following transfer, remove the membrane from the blotting cassette and mark the orientation of the gel with a pencil. Rinse briefly with PBS.
*Transfer Buffer:
Tris base 48.5 g Glycine 240.2 g Methanol 3.2 L 10N NaOH 1.5 ml water to 16 L
Antibody Detection:
Wash the membrane with TBST* once for 5 min at room temperature. Block non-specific binding on the membrane with freshly prepared 5% nonfat dried milk for 1 hour on a shaking platform at room temperature.
Wash three times for 5 minutes each with TBST. Incubate the membrane with a specific primary antibody diluted in TBST and 5% BSA at the manufacturer’s recommended dilution with gentle agitation at 4o C overnight.
Wash three times for 5 min each with TBST.
Incubate with OriGene's HRP-conjugated secondary antibody (TA100015) at 1:20,000 in TBST-5% BSA for 1 hour at room temperature.
Wash three times again for 5 minutes each with TBST.
For detection, use OriGene's Western Blot Luminol Reagent (TA100016) and prepare according to instructions.
Lay the membrane on a plastic surface with the protein side up. Add the mixed detection solution to the membrane. Incubate for 1 minute. Remove the excess solution and cover the membrane with transparent plastic.
Place the wrapped blot with protein side up in an X-ray film cassette. Place a sheet of X-ray autoradiography film on the top of the membrane. Close the cassette for 15 sec to 1 min. Remove the film for development. Add additional films if needed for longer or shorter exposures.
