Anti-RCC2 抗体 (ab70787)

Thank you for contacting us. Because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

If the product is to be used in an untested species or application, you may be eligible for our AbTrial program if the antibody has not yet been purchased. Please contact our Scientific Support team by replying to this email prior to purchase for more information. Or you can find more information about this program at the following link:

www.abcam.com/abtrial

Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

To find out more about our Abreview system, please see the following link:

https://www.abcam.com/abreviews

I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

Thank you for your enquiry. I am pleased to provide the following testing protocol for ab70787 RCC2 antibody in IP. Preparation of Cell Lysate used for Immunoprecipitation Reagents needed: PBS (Phosphate Buffered Saline, pH 7.2) NaCl 8.0 g KCl 0.2 g NaH2PO4 0.23 g Na2HPO4 0.12 g Adjust the volume to 1 liter with distilled water. Store at 4-25 C. IP Lysis Buffer NaCl 7.31 g 1 M Tris, pH 8 25 ml 0.5 M EDTA, pH 8 5 ml 10% NP-40 25 ml Distilled Water 445 ml Store at 4 C. Protease Inhibitors (Calbiochem Cat#539131) Reconstitute with 1 ml of dH2O. Use 50 mcl per 5 ml of lysis buffer to harvest cells. Procedure: 1. Allow cells to grow to approximately 80% confluence on 100mm polystyrene tissue culture plates. Approximately 8*106 cells (one plate at 80% confluence) are needed per IP reaction. 2. Wash cells two times in cold PBS. 3, Lyse cells to release soluble cellular proteins using 500 mcl of cold lysis buffer containing 1X protease inhibitors (Calbiochem, Cat. No. 539131) per 100 mm plate. 4. Scrape cells from the plates, transfer to 1.5 ml micro-centrifuge tubes, and place on ice for 30 minutes to ensure efficient lysis. 5. Centrifuge lysates at 10,000 x g; 5 minutes. 6. Remove and pool the supernatant. Determine protein concentration. Immunoprecipitation Protocol Reagents Needed: Immunoprecipitation (IP) lysis buffer Protease Inhibitors Primary Antibodies made in Rabbit Normal IgG, negative control Protein A Sepharose Beads Cell Lysate Sample Buffer IP lysis Buffer 12.5 ml 1M NaCl (250mM) 2.5 ml 1M Tris (50mM) 500ul 0.5M EDTA (5mM) 2.5ml 10% NP-40 32 ml dH20 Protein A Beads Resuspend 400 mg of Protein A beads in 10 ml of distilled H2O. Mix well to resuspend. Spin at 250 rpm for 5 minutes. Wash 3X in 10 ml IP Lysis buffer. Resuspend to 10 ml with IP lysis buffer for a 20% solution. Use 100 mcl per IP reaction. 4X Sample Buffer Glycerol 4.0 g Tris Base 0.68 g Tris HCL 0.67 g LDS 0.80 g EDTA 6 mg Brilliant Blue G250 2.5 mg Phenol red 2.5 mg Adjust volume to 10 ml with ultra pure water. Store at 4 C. 4X sample buffer is available from Invitrogen (Cat# NP0007) 1X Sample Buffer 4X sample buffer 150 ml 1M DTT 60 ml Distilled water 390 ml Make fresh for each use. Procedure: 1. Prepare cell lysate according to protocol. Place 500 mcl of the prepared cell lysate (1-3 mg/ml) into a 1.5 ml micro-centrifuge tube. 2. To this tube add 2 to 10 mcg of the primary antibody (If using neat sera or an IgG fraction such as Protein-A purified antibody, larger amounts are likely to be required. For best results, optimal amounts of antibody should be empirically defined.) 3. To a negative control reaction, add an equivalent amount of normal rabbit IgG. 4. Add 100 ml of a 20% Protein A suspension to the mixture of antibody and cell lysate. 5. Rotate the immunoprecipitation reactions (end-to-end) for 3 hours at room temperature or overnight at 4 C. 6. Centrifuge (200 x g; 5 minutes) to pellet the complex. Remove the supernatant and add 500 ml cold cell lysis buffer. Centrifuge (200 x g; 5 minutes). 7. Repeat wash step 6 twice more. After each centrifugation remove as much of the supernatant as possible. 8. After removing the supernatant from the third wash, add 40 mcl of freshly prepared 1X sample buffer to each tube and heat at 90 C for 5 minutes. 9. Continue with electrophoresis and immunoblotting. Load 8 to 16 ml (20 to 40% of the IP reaction) to a polyacrylamide gel. Note: For optimal results, complete reduction of the sample is required. We recommend the use of 0.1 M DTT in SDS-PAGE sample buffer and immediately heating samples, loading and running gels. I hope this will be helpful to you. Should you require anything further, please do not hesitate to contact us.