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.
Recommended Dilution:Western Blot: detects activated Erb B2/HER-2 in RIPA lysates from heregulin stimulated MCF-7 breast carcinoma cells.
Lysates from non-stimulated and heregulin-stimulated MCF-7 cells were resolved by electrophoresis, transferred to nitrocellulose and probed with E3451-25 (1:2000 dilution).
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system
Optimal dilution determined by the researcher.
Immunoblot Protocol 1.
Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a cell lysate sample (cell lysis buffer: 50mM Tris-HCl, pH7.4; 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 PBS containing 3% nonfat dry milk (PBS-MLK) for 30 minutes at RT with constant agitation.
3.
Incubate the nitrocellulose with a 1:2000-1:8000 dilution of E3451-25 diluted in freshly prepared PBS-MLK overnight with agitation at 4°C.
4.
Wash the nitrocellulose twice with water.
5.
Incubate the nitrocellulose in the secondary reagent of choice (a goat anti-rabbit HRP conjugated IgG, 1:5000 dilution was used) in PBS-MLK for 1.5 hours at room temperature with agitation.
6.
Wash the nitrocellulose with water twice.
7.
Wash the nitrocellulose in PBS-0.05% Tween 20 for 3-15 minutes.
8.
Rinse the nitrocellulose in water for 30-60min.
9.
Use detection method of choice (enhanced chemiluminescence was used).
Storage and Stability:Store product at 4°C in the dark.
DO NOT FREEZE! Stable at 4°C for 12 months after receipt as an undiluted liquid.
Dilute required amount only prior to immediate use.
Further dilutions can be made in assay buffer.
Caution: MaxLight™650 conjugates are sensitive to light.
For maximum recovery of product, centrifuge the original vial prior to removing the cap.
Note: Applications are based on unconjugated antibody.
仕様
Size：100ul
Host：rabbit
Source Antibody：human
Grade：Affinity Purified
Purity：Purified by immunoaffinity chromatography.
Form：Supplied as a liquid in PBS, pH 7.2. No preservative added. Labeled with MaxLight™650.
Specificity：Reecognizes the C-terminus of human erbB-2/HER-2 phosphorylated at Tyr1248.
Isotype：IgG
Calc Applications Abbrev：WB
Calc Crossreactivity：Hu
Immunogen：Synthetic phosphopeptide, residues 1243-1255, corresponding to a major auto-phosphorylation (Tyr1248) site of human erbB-2/HER-2 (C-AENPEpYLGLDVPV). Species sequence homology (100%): mouse, rat and monkey
