NCOA4 antibody (N-Term)

Catalog No. ABIN2780237

Product Details anti-NCOA4 Antibody

Target: NCOA4 (Nuclear Receptor Coactivator 4 (NCOA4))

Binding Specificity: N-Term

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

Host: Rabbit

Clonality: Polyclonal

Conjugate: This NCOA4 antibody is un-conjugated

Application: Western Blotting (WB)

Sequence: CLIHQLEYTQ NKDLANQVSV CLERLGSLAL KPEDSTVLLF EADTSALRQT

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

Characteristics: This is a rabbit polyclonal antibody against Ncoa4. It was validated on Western Blot.

Purification: Affinity Purified

Immunogen: The immunogen is a synthetic peptide corresponding to a region of Mouse

Application Details

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

Comment:

Antigen size: 625 AA

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100057 (Western Blotting)

by: University of Michigan Medical School, Molecular & Integrative Physiology, Shah lab

No.: #100057

Date: 09/12/2016

Antigen: Ncoa4

Lot Number: QC11327

Method validated: Western Blotting

Positive Control: HIF overexpression mice, WT mice

Negative Control: NCOA4-/- mice

Notes: Passed, based on the fact that the antibody picked up the positive control (unpublished data suggests that NCOA4 is highly expressed in an intestinal HIF overexpression model).

Validation Images

Mouse duodenum whole cell lysates from intestinal HIF overexpression (HIF OE) mice, NCOA4-/- mice, and wt mice fed a normal or low iron diet were separated by SDS-PAGE. NCOA4 was visualized using ABIN2780237. A ferritin (Ftn) Western blot was done to confirm the efficacy of iron diet regimens in mice. GAPDH served as loading control.

Full Methods

Primary Antibody: ABIN2780237

Secondary Antibody: Anti-rabbit HRP-linked secondary antibody (Cell Signaling, 7074S, lot 26)

Full Protocol:

• Wild type (WT) or NCOA4 -/- mice were fed normal or iron-deficient diet for 7 days, followed by euthanization and scraping of duodenal epithelium (DE).
• DE cells were lysed in RIPA buffer and whole cell lysate (WCL) prepared.
• 50µg total protein from each sample were used for Western blot analysis.
• Samples were denatured in 5x gel-loading buffer containing beta-marcaptoethanol and separated on a 10% SDS-PAGE gel (Laemmli 1970).
• Transfer onto Nitrocellulose membrane (Biorad, 1620212, lot 10484060) was done on Trans-Blot SD apparatus (Bio-Rad) following the manufacturer’s protocol.
• Membranes were blocked with TBS-tween (TBST) solution containing 5% milk for 1h.
• Incubation with primary antibody overnight at 4°C:
• • NCOA4 antibody ABIN2780237 (lot QC11327) diluted 1:1000 in TBST containing 0.1% BSA.
  • FTH1 antibody (Cell signaling, 3998S; lot 1) diluted in TBST containing 0.1% BSA.
  • GAPDH antibody (Santa Cruz, SC-25778, lot D30115) diluted in TBST containing 0.1% BSA.
• Washing with TBST for 15min at RT.
• Incubation with anti-rabbit HRP-linked secondary antibody (Cell Signaling, 7074S, lot 26) diluted 1:2000 in TBST a shaker for 1h at RT.
• Three rinses, followed by four 15min washes, twice TBST and twice with TBS.
• Chemiluminescence detection was done using ECL Select Western blotting detection reagent (Amersham, RPN2235, lot 9723881).
• Blot was developed on a Biorad imaging system.

Experimental Notes:

• The antibody did not consistently pick up the WT mice on normal and low iron diet.
• Also, there is still a faint band in the NCOA4 -/- lane. We are confident that it is a true KO mouse, as there is high ferritin expression even after 7 days of low iron diet (compared to ferritin expression in WT low iron mice as well as that of the HIF over expression mouse, which are known to have high intestinal ftn content).

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 NCOA4

Target: NCOA4 (Nuclear Receptor Coactivator 4 (NCOA4))

Alternative Name: Ncoa4 (NCOA4 Products)

Synonyms: zgc:55307 antibody, zgc:77270 antibody, NCOA4 antibody, MGC138109 antibody, rfg antibody, ele1 antibody, ptc3 antibody, ara70 antibody, MGC84996 antibody, DKFZp469A035 antibody, DKFZp459B2429 antibody, DKFZp469A1925 antibody, ncoa4 antibody, ARA70 antibody, ELE1 antibody, PTC3 antibody, RFG antibody, 1110034E15Rik antibody, 4432406M01Rik antibody, AI227008 antibody, Rfg antibody, nuclear receptor coactivator 4 antibody, nuclear receptor coactivator 4 S homeolog antibody, Ncoa4 antibody, ncoa4 antibody, NCOA4 antibody, ncoa4.S antibody

Background: The function of Ncoa4 remains unknown.
Alias Symbols: 1110034E15Rik, 4432406M01Rik, AI227008, ARA70, MGC103382, Rfg
Protein Interaction Partner: Ndn,
Protein Size: 625

Molecular Weight: 70 kDa

Gene ID: 27057

NCBI Accession: NM_001033988, NP_001029160

Pathways: Intracellular Steroid Hormone Receptor Signaling Pathway