ACVRL1 Rabbit Recombinant mAb | Monoclonal Antibodies

ACVRL1 Rabbit Recombinant mAb

Catalog No.A5397

For research use only.

Advantages

1. Why choose rabbit monoclonal antibody?
Rabbit monoclonal antibody has over 100 times higher affinity than that of mouse monoclonal.

2.Why choose recombinant antibodies?
Recombinant antibodies are known for higher purity and minimal deviation between batches, versus regular antibodies.

Application Data

WB

Validated by Selleck

IHC

Validated by Selleck

Usage Information

Application WB, IHC,ELISA
Dilution
WB IHC
1:1000 1:50
Reactivity Human Mouse Rat
MW (kDa) 56kDa
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.

IHC

Immunohistochemistry (Paraffin)

Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

1. Deparaffinize/hydrate sections:

1. Incubate sections in three washes of xylene for 5 min each.
2. Incubate sections in two washes of 100% ethanol for 10 min each.
3. Incubate sections in two washes of 95% ethanol for 10 min each.

2. Wash sections two times in dH2O for 5 min each.

Antigen retrieval

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections:
1. Incubate sections in 95% ethanol two times for 10 sec each.
2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
3. Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.

Description

Specificity ACVRL1 Rabbit Recombinant mAb detects endogenous level of total ACVRL1.
Background ACVRL1 (Serine/threonine-protein kinase receptor R3, activin receptor-like kinase 1 and ALK1) is a receptor in the TGF beta signaling pathway. Activation of ALK1 results from the binding of several different ligands of the TGF-β family, including bone morphogenetic protein (BMP) 9, BMP10, and TGF-β. ALK1 is predominantly expressed in endothelial cells and at specific sites of epithelial-mesenchymal interactions, stimulates Smad1 and Smad5 phosphorylation. The TGFβ/ALK5 and TGFβ/ALK1 pathways have opposite effects on endothelial cells (ECs) behavior; ALK5 inhibits EC migration and proliferation while ALK1 stimulates both processes. ALK1 is a negative mediator of lateral TGFβ/ALK5 signaling and its optimal activation requires ALK5 kinase activity. TGFβ regulates cellular processes by binding to a heteromeric complex of type I and type II serine/threonine kinase receptors. In most cell types, TGFβ initially binds TβRII and subsequently recruits and phosphorylates ALK5. Activated ALK5 propagates the signal into the nucleus by inducing the phosphorylation of Smad2 and Smad3. In endothelial cells, however, TGFβ can signal via two distinct type I receptor/Smad pathways. Whereas activation of ALK5/Smad2/3 pathway by TGFβ results in inhibition of migration and proliferation, TGFβ-induced ALK1/Smad1/5 activation results in increased migration and proliferation. ALK1 not only induced biological responses opposite from those of ALK5, but also directly inhibited ALK5/Smad signaling.

Datasheet & SDS

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