Mouse Anti-Raf1 (S338), phosphorylated (MaxLight 650)
MaxLight™650 is a new Far-IR stable dye conjugate comparable to Alexa Fluor™647, DyLight™649, Cy5™ and offers better labeling efficiency, brighter imaging and increased immunodetection. Absorbance (655nm); Emission (676nm); Extinction Coefficient 250,000.
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.
Note: Applications are based on unconjugated antibody.
Immunogen
Synthetic peptide around the region of phosphorylated Ser338 of human Raf-1; conjugated to KLH. Species sequence homology: rat and chicken -100%, Xenopus-92%.
Form
Supplied as a liquid in PBS, pH 7.2. No preservative added. Labeled with MaxLight™650.
Purity
Purified by Protein G affinity chromatography.
Specificity
Recognizes phosphorylated human Raf-1 (S338), Mr 74kD. Species crossreactivity: Mouse
