|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||GluR2 Rabbit Recombinant mAb detects endogenous level of total GluR2.|
|Background||In the mammalian central nervous system, AMPA-type glutamate receptors mediate the vast majority of fast excitatory synaptic transmission. AMPARs are tetramers made up of combinations of four subunits: GluR1, GluR2, GluR3, and GluR4. AMPARs are widely expressed throughout the central nervous system both in neurons and in glia. The great majority of AMPARs in the central nervous system exist as heteromers containing GluR2. During early postnatal development, expression of GluR2 is low compared with that of GluR1, but it increases rapidly during the first postnatal week. GluR2 is a critical subunit in determining mammalian AMPAR function. This subunit determines many of the major biophysical properties of the native receptor, including, but not limited to, receptor kinetics, single-channel conductance, Ca2+ permeability, and block by endogenous polyamines. In addition, it is the most tightly regulated of the glutamate receptor subunits, with a number of specific regulatory processes at the level of gene expression, RNA editing, receptor assembly, and trafficking. It is of critical importance for normal brain function. GluR2 subunit dictates the critical biophysical properties of the receptor, strongly influences receptor assembly and trafficking, and plays pivotal roles in a number of forms of long-term synaptic plasticity.|