|Reactivity||Human Mouse Rat|
|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||Hemoglobin subunit alpha Rabbit Recombinant mAb detects endogenous level of total Hemoglobin subunit alpha.|
|Background||Hemoglobin is the major oxygen carrying component in blood and is vital to the survival of most multicellular animals. In higher order vertebrates, this essential protein is a tetramer composed of two related though distinct globin chains: alpha-globin and beta-globin. It is crucial to erythrocyte formation that the two globin chains are produced at balanced levels. Any disruptions resulting in an excess of either chain has significant, deleterious effects on red cell survival. haem binding stabilizes the globin chains and promote proper folding of the polypeptide. haem is essential to the function of Hemoglobin. α-globin has a ten-fold greater ability to bind haem compared to β-globin and may actually facilitate haem integration into apo-β-globin (and γ-globin) chains. As free α-globin is an unstable, reactive molecule capable of destroying erythroid progenitor cells, this small pool of excess α-globin chains must be stabilized to limit its reactivity. This is achieved by the binding of an abundant erythroid-expressed protein known as α-hemoglobin stabilizing protein (AHSP). AHSP is able to interact with multiple forms of α-globin including apo-, ferrous and ferric states bound to a variety of ligands and reduces oxidant-induced precipitation of α-globin in solution. AHSP binds α-globin at the α1β1 dimer interface, opposite the haem binding pocket, with lower affinity than β-globin and is easily displaced by β-globin binding.|