JNK1/JNK3 Rabbit Recombinant mAb | Monoclonal Antibodies

JNK1/JNK3 Rabbit Recombinant mAb

Catalog No.A5026

For research use only.

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  • WB

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Usage Information

Application WB,ELISA
Dilution
WB
1:1000
Reactivity Human Mouse Rat
MW (kDa) 46,54kDa
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

JNK1/JNK3 Rabbit Recombinant mAb detects endogenous levels of total JNK1/JNK3.

Background

JNKs are stress-activated protein kinases (SAPKs) which play a key role in the responses stimulated by proin-ammatory cytokines, environmental stress, and apoptotic agents. The c-Jun N-terminal kinases consist of ten isoforms derived from three genes: JNK1 (four isoforms), JNK2 (four isoforms) and JNK3 (two isoforms). Each gene is expressed as either 46 kDa or 55 kDa protein kinases, depending upon how the 3' coding region of the corresponding mRNA is processed. There have been no functional differences documented between the 46 kDa and the 55 kDa isoform, however, a second form of alternative splicing occurs within transcripts of JNK1 and JNK2, yielding JNK1-α, JNK2-α and JNK1-β and JNK2-β. Differences in interactions with protein substrates arise because of the mutually exclusive utilization of two exons within the kinase domain. JNK1 and JNK2 are found in all cells and tissues. JNK3 is found mainly in the brain, but is also found in the heart and the testes.

Datasheet & SDS

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