GCLC Rabbit Recombinant mAb | Monoclonal Antibodies

GCLC Rabbit Recombinant mAb

Catalog No.A5306

For research use only.

Filter:

  • WB

Usage Information

Application WB,ELISA
Dilution
WB
1:1000
Reactivity Human Rat
MW (kDa) 73kDa
Source Rabbit
Concentration 1mg/ml
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.

Protocol

WB

Western Blotting

Sample preparation

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

a. Blocking

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.

Description

Specificity GCLC Rabbit Recombinant mAb detects endogenous level of total GCLC.
Background Glutamate cysteine ligase (GCL) is a holoenzyme comprising a ∼73 kDa catalytic subunit (GCLC), 637 amino acids in length and a ∼31 kDa modifier (GCLM) subunit, 274 amino acids in length. The specific activity of GCL is a major determinant of the rate of GSH (glutathione) synthesis and homeostasis. GCLC (Glutamate-cysteine ligase catalytic subunit) is the first rate limiting enzyme of glutathione synthesis. The enzyme consists of two subunits, a heavy catalytic subunit and a light regulatory subunit. The GCLC active site facilitates the formation of the peptide bond between cysteine's amino group and glutamate's γ-carboxyl group.

Datasheet & SDS

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