Smad4 Rabbit Recombinant mAb

Catalog No.A5130

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  1. 1. Why choose rabbit monoclonal antibody?
  2. Rabbit monoclonal antibody has over 100 times higher affinity than that of mouse monoclonal.
  3. 2. Why choose recombinant antibodies?
  4. Recombinant antibodies are known for higher purity and minimal deviation between batches, versus regular antibodies.

WB

Validated by Selleck

Usage Information

Application WB,ELISA
Dilution
WB
1:1000
Reactivity Human Mouse Rat
MW (kDa) 60kDa
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
+ Expand

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 Smad4 Rabbit Recombinant mAb detects endogenous level of total Smad4.
Background Transforming growth factor-β (TGF-β) family members exert their function via specific type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors, including the common mediator Smad4. Smad4 is the pivotal factor of the TGF-β pathway and functions as a key tumor suppressor. Smad4-mediated cellular responses to TGF-β signaling vary with extracellular matrix, ligand concentration, and cell type specific cofactors at different developmental stages. Upon ligand binding, TGF-βR phosphorylates and activates receptor-associated Smad2 and Smad3, which subsequently associate with a co-Smad, Smad4, to control target gene expression. Smad4 is largely dispensable for T cell activation and Th cell differentiation, it is required for T cell proliferation in response to cognate antigen to promote T-cell-mediated inflammatory disease and tumor rejection.

Datasheet & MSDS

TGF-beta/Smad Signaling Pathway Map

Related TGF-beta/Smad Products

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