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 if to be used immediately within two weeks.
For long-term storage, aliquot to avoid repeated freezing and thawing and store at -20°C.
Aliquots are stable at -20°C for 12 months after receipt.
Dilute required amount only prior to immediate use.
Further dilutions can be made in assay buffer.
For maximum recovery of product, centrifuge the original vial after thawing and 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 Biotin.
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
