|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.
|Specificity||L1CAM Rabbit Recombinant mAb detects endogenous level of total L1CAM.|
|Background||L1CAM is a cell adhesion molecule of the immunoglobulin superfamily involved in the development of the nervous system and progression of malignancies. The 200-220 kDa transmembrane glycoprotein consists of a conserved cytoplasmic part, five fibronectin type III repeats and six immunoglobulin-like (Ig) domains. Ig-like domains are very efficient in recognizing and binding specific partners, play a major role in homophilic interactions between adjacent cells and are involved in a number of heterophilic interactions. The L1CAM fibronectin type III (FN) modules I-V were shown to directly interact with the immunoglobulin modules of fibroblast growth factor receptor 1 (FGFR1) in an ATP-dependent manner and this interaction plays an important role in modulating neuronal differentiation and FGFR1 phosphorylation. The cytoplasmic part of L1CAM can serve as a scaffold for the assembly of cytoskeleton components.|