ARHGEF4 antibody (N-Term)

Catalog No. ABIN2787993

This anti-ARHGEF4 antibody is a Rabbit Polyclonal antibody detecting ARHGEF4 in WB. Suitable for Human, Mouse, Rat, Cow, Dog, Guinea Pig, Horse and Rabbit. Independently validated for use in Western Blotting.

Quick Overview for ARHGEF4 antibody (N-Term) (ABIN2787993)

Target: ARHGEF4 (rho Guanine Nucleotide Exchange Factor (GEF) 4 (ARHGEF4))

Reactivity: Human, Mouse, Rat, Cow, Dog, Guinea Pig, Horse, Rabbit

Host: Rabbit

Clonality: Polyclonal

Conjugate: This ARHGEF4 antibody is un-conjugated

Application: Western Blotting (WB)

Product Details anti-ARHGEF4 Antibody

Binding Specificity: N-Term

Sequence: ADDYEAPRAG AREADDSGPE AQSCKDQMRT NVINEILSTE RDYIKHLRDI

Predicted Reactivity: Cow: 79%, Dog: 93%, Guinea Pig: 92%, Horse: 86%, Human: 100%, Mouse: 86%, Rabbit: 100%, Rat: 100%

Characteristics: This is a rabbit polyclonal antibody against Arhgef4. It was validated on Western Blot.

Purification: Affinity Purified

Immunogen: The immunogen is a synthetic peptide directed towards the N-terminal region of Mouse Arhgef4

Application Details

Application Notes: Optimal working dilutions should be determined experimentally by the investigator.

Comment:

Antigen size: 484 AA

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100106 (Western Blotting)

by: Instituto de Parasitología y Biomedicina López-Neyra

No.: #100106

Date: 12/23/2016

Antigen: ARHGEF4

Lot Number: QC30159-41213

Method validated: Western Blotting

Positive Control: primary mouse microglia

Notes: Passed. ABIN2787993 specifically recognizes Arhgef4 in primary mouse microglia lysates.

Validation Images

Indicated amounts of total protein from primary mouse microglia cell lysate were separated on a denaturing SDS-PAGE gel and incubated with ABIN2787993 as described in the protocol.

Full Methods

Primary Antibody: ABIN2787993

Secondary Antibody: anti-rabbit HRP-conjugated antibodies (DakoCytomation, P0448, lot 00055814)

Full Protocol:

• Primary mouse microglia are grown in DMEM (Invitrogen), supplemented with 10% foetal bovine serum (Gibco), 10% horse serum and penicillin and streptomycin (Gibco), at 37°C and 5% CO2 in a 12-well dish.
• Lyse cells in 25µl per well cold lysis buffer (10mM Tris-HCl pH 8.0, 150mM NaCl, 1% Nonidet-P40, 1mM EDTA, 10mM NaF, 1mM Na₃VO₄, protease inhibitors (Sigma)).
• Determine total protein content of the lysates using Bradford assay (Bio-Rad, 500-0006, lot 111832).
• Denature 5µg, 10µg and 30µg total protein for 5min at 95°C in 5µl, 10µl and 20µl Laemmli SDS sample buffer and subsequently separate them on a denaturing self-made 10% SDS-PAGE for 1h at 140V.
• Transfer proteins onto PVDF membrane (Pall Life Sciences, 75696G, lot TO3225) with a semi-dry Western blotting system for 50min at 60mA.
• Block the membrane with TBST (TBS, 0.1% Tween) containing 5% milk for 1h at RT.
• Wash membrane 3x 5min with TBST.
• Incubation with primary rabbit anti-ARHGEF4 antibody (antibodies-online, ABIN2787993, lot QC30159-41213) diluted 1:500 in TBST containing 2% BSA ON at 4°C.
• Wash membrane 5x 5min with TBST.
• Incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, P0448, lot 00055814) diluted 1:2000 in TBST containing 5% milk for 1h at RT.
• Wash membrane 5x 5min with TBST and 3x 5min with TBS.
• Reveal protein bands using ECL Plus Western Blotting Detection Reagent (GE Healthcare, RPN2132, lot 4645641) with Curix RP2 Plus medical X-ray films (AGFA Health Care, ENKMV, lot 79040074) on an AGFA Health Care Curix60-developer.

Experimental Notes: The ARHGEF4 antibody ABIN2787993 reveals multiple bands as is expected for murine Arhgef4.

Handling

Format: Liquid

Concentration: Lot specific

Buffer: Liquid. Purified antibody supplied in 1x PBS buffer with 0.09 % (w/v) sodium azide and 2 % sucrose.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

Storage Comment: For short term use, store at 2-8°C up to 1 week. For long term storage, store at -20°C in small aliquots to prevent freeze-thaw cycles.

Target Details for ARHGEF4

Target: ARHGEF4 (rho Guanine Nucleotide Exchange Factor (GEF) 4 (ARHGEF4))

Alternative Name: Arhgef4

Background: Arhgef4 acts as guanine nucleotide exchange factor (GEF) for RHOA, RAC1 and CDC42 GTPases. Binding of APC may activate RAC1 GEF activity. The APC-ARHGEF4 complex seems to be involved in cell migration as well as in E-cadherin-mediated cell-cell adhesion By similarity. It is required for MMP9 up-regulation via the JNK signaling pathway in colorectal tumor cells. Arhgef4 is involved in tumor angiogenesis and may play a role in intestinal adenoma formation and tumor progression.
Alias Symbols: 9330140K16Rik, Asef, ENSMUSG00000070955
Protein Size: 484

Molecular Weight: 57 kDa

Gene ID: 226970

NCBI Accession: NM_183019, NP_898840

UniProt: Q7TNR9

Pathways: Neurotrophin Signaling Pathway