|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||Lamin A/C Rabbit Recombinant mAb detects endogenous levels of total Lamin A/C.|
|Background||The lamins are the major architectural proteins of the animal cell nucleus. Lamins line the inside of the nuclear membrane, where they provide a platform for the binding of proteins and chromatin and confer mechanical stability. They have been implicated in a wide range of nuclear functions, including higher-order genome organization, chromatin regulation, transcription, DNA replication and DNA repair. The lamins are members of the intermediate filament (IF) family of proteins, which constitute a major component of the cytoskeleton. Two major kinds of lamins are present in mammalian cells: B-type lamins (B1 and B2), which are found in nearly all somatic cells, and A-type lamins (A and C), which are expressed only in differentiated cells. Nucleoplasmic lamin A/C is highly mobile. Lamin A/C associates with numerous transcriptional regulators, either directly or indirectly, including Rb, Gcl, Mok2, cFos and Srebp1.|