Anti-phospho-Raf-1 (Ser338), clone 1.T.16

Applications: , Suitable for use in Western Blot. Other applications have not been tested. Recommended Dilution: Western Blot: 0.5-2ug/ml detects phosphorylated Raf-1(S338) in RIPA lysates from 3T3 cells stimulated with 50ng/ml PDGF for 20 minutes. An unstimulated and PDGF-stimulated (50ng/ml, 20 minutes, 3T3 cell lysate were resolved by electrophoresis, transferred to nitrocellulose and probed with R0495-03 (2ug/ml). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a chemiluminescence detection system., Optimal dilutions to be determined by the researcher. Western Blot Protocol 1. Perform SDS-PAGE on a cell lysate sample (cell lysis buffer: 50mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150mM NaCl, 1mM EGTA, 1mM PMSF, 1ug/ml each aprotinin, leupeptin, pepstatin, 1mM Na3VO4, 1mM NaF) and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water. 2. Block the blotted nitrocellulose in freshly prepared TBS containing 0.05% Tween-20 and 5% nonfat dry milk (TBST-MLK) for 1 hour at RT with constant agitation. 3. Incubate the nitrocellulose with 0.5-2ug/ml R0495-03, diluted in freshly prepared TBST-MLK overnight with agitation at 4°C. 4. Wash the nitrocellulose twice with water. 5. Incubate the nitrocellulose in the secondary reagent of choice (e.g., a goat anti-mouse HRP conjugated IgG) in TBST-MLK for 1.5 hours at RT with agitation. 6. Wash the nitrocellulose with water twice. 7. Wash the nitrocellulose in TBS-0.1% Tween 20 for 3-5 minutes. 8. Rinse the nitrocellulose in 4-5 changes of water. 9. Use detection method of choice (enhanced chemiluminescence was used). Storage and Stability: May be stored at 4°C for short-term only. For long-term storage, aliquot to avoid repeated freezing and thawing and freeze at -20°C. Aliquots are stable for 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.