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Western blot analysis of alpha-fetoprotein was performed by loading 10 ug of whole cell extract from H9 ESCs, H9 ESC-derived hepatoblasts, ESC-derived hepatocyte-like cells, HepG2 cells, HepaRG cells, and primary human hepatocytes in reducing conditions and 10 ul Spectra™ Multicolor Broad Range Protein Ladder (Product # 26634) per well onto a Novex Bolt® 4-12% Bis-Tris Plus Gel with Bolt® MOPS running buffer and Bolt® Antioxidant. Proteins were transferred to a PVDF membrane using the iBlot 2 Dry Blotting System (Product # IB21001), and blocked with 5% non-fat milk in TBST for 1 hour at room temperature. alpha-Fetoprotein was detected at 67 kDa using an alpha-fetoprotein monoclonal antibody (Product # MA5-14666) at a concentration of 1 ug/ml in 5% non-fat milk in TBST overnight at 4°C on a rocking platform, followed by a goat anti-mouse secondary antibody (Product # A24518) at a dilution of 1:10,000 for 1 hour at room temperature. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Mouse|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Storage buffer||PBS, pH 7.2, with 1% BSA|
|Contains||0.1% Kathon™ CG|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Cross Adsorption||Against bovine, goat, human, rabbit and rat IgG|
|Antibody Form||F(ab')2 Fragment|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:500-1:20,000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Anti-Mouse secondary antibodies are affinity-purified antibodies with well-characterized specificity for mouse immunoglobulins and are useful in the detection, sorting or purification of its specified target. Secondary antibodies offer increased versatility enabling users to use many detection systems (e.g. HRP, AP, fluorescence). They can also provide greater sensitivity through signal amplification as multiple secondary antibodies can bind to a single primary antibody. Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (i.e. immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.