|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
NOTE: Do not allow slides to dry at any time during this procedure.
1. Deparaffinize/hydrate sections:
1. Incubate sections in three washes of xylene for 5 min each.
2. Wash sections two times in dH2O for 5 min each.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
1. Wash sections in dH2O three times for 5 min each.
|Specificity||Cytokeratin 18 Rabbit Recombinant mAb detects endogenous level of cytokeratin 18.|
|Background||The intermediate filaments consist of a large number of nuclear and cytoplasmic proteins that are expressed in a tissue- and differentiation-dependent manner. The components of the intermediate filaments are highly conserved during evolution and show a high degree of conservation among species. Cytokeratins are major structural proteins found in epithelial cells, which form the cytoplasmic network of intermediate filaments. Cytokeratins consist of at least 20 unique gene products that fall into two categories: the relatively acidic type I group (CK9-CK20) and the neutral-basic type II group (CK1-CK8). Cytokeratin 18 (CK18) is a type I intermediate filament protein that is found primarily in many types of single-layered or "simple" epithelial tissues and is localized in the cytoplasm and perinuclear region. CK18 and its coexpressed complementary type II keratin partner, CK8, are persistently expressed in a variety of adult epithelial organs, such as the liver, lung, kidney, pancreas, gastrointestinal tract, and mammary gland, and are also expressed by cancers that arise from these tissues. The known function of CK18 is to provide a flexible intracellular scaffolding to structure cytoplasm, resist stresses externally applied to the cell and maintain normal mitochondrial structures. CK18 is also important for cellular processes such as apoptosis, mitosis, cell cycle progression, and cell signaling.|