RHOT1 antibody (N-Term)

Catalog No. ABIN405704

The Rabbit Polyclonal anti-RHOT1 antibody has been validated for WB and has been independently validated. It is suitable to detect RHOT1 in samples from Human, Mouse, Rat, Dog, Cow, Guinea Pig, Horse, Rabbit and Zebrafish (Danio rerio).

Quick Overview for RHOT1 antibody (N-Term) (ABIN405704)

Target: RHOT1 (Ras Homolog Gene Family, Member T1 (RHOT1))

Reactivity: Human, Mouse, Rat, Dog, Cow, Guinea Pig, Horse, Rabbit, Zebrafish (Danio rerio)

Host: Rabbit

Clonality: Polyclonal

Conjugate: This RHOT1 antibody is un-conjugated

Application: Western Blotting (WB)

Product Details anti-RHOT1 Antibody

Binding Specificity: N-Term

Sequence: EMKPACIKAL TRIFKISDQD NDGTLNDAEL NFFQRICFNT PLAPQALEDV

Predicted Reactivity: Cow: 100%, Dog: 100%, Guinea Pig: 100%, Horse: 100%, Human: 100%, Mouse: 100%, Rabbit: 100%, Rat: 100%, Zebrafish: 85%

Characteristics: This is a rabbit polyclonal antibody against RHOT1. It was validated on Western Blot using a cell lysate as a positive control.

Purification: Affinity Purified

Immunogen: The immunogen is a synthetic peptide directed towards the N terminal region of human RHOT1

Application Details

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

Comment:

Antigen size: 659 AA

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100008 (Western Blotting)

by: Shaw Lab, Department of Biochemistry, University of Utah

No.: #100008

Date: 09/12/2015

Antigen: RHOT1

Method validated: Western Blotting

Positive Control:

Purified recombinant Miro1

Isolated mitochondria from immortalized Miro1 KO MEFs expressing Myc-Miro1

Negative Control:

Purified recombinant Miro2

Isolated mitochondria from immortalized Miro1 WT MEFs

Isolated mitochondria from immortalized Miro1 KO MEFs expressing Myc-Miro2

Notes:

Passed. ABIN405704 specifically recognizes Miro1 without cross-reaction with Miro2.

Validation Images

Full Methods

Primary Antibody: ABIN405704

Secondary Antibody: Goat-anti-rabbit IgG HRP conjugated (Sigma, A0545-1M22)

Full Protocol:

• Transfect immortalized Miro1-/- MEFs with Myc-Miro1-V1 or Myc-Miro2-V1 as described in PMID 25136135.
• Isolate mitochondria from immortalized Miro1+/+, Miro1-/-, Miro1-/- + Myc-Miro1 and Miro1-/- + Myc-Miro2 MEFs.
• Determine protein concentration and snap freeze mitochondria in liquid nitrogen.
• Thaw 30 µg mitochondria per lane on ice.
• Thaw 100 ng recombinant Miro1 and Miro2 per lane on ice.
• Add 2x Laemmli buffer and 2% BME to samples and denature for 5 min at 95 °C.
• Separte 30 µg/lane mitochondria and 100 ng/lane recombinant protein on a 12.5% SDS-PAGE for 3 h at 40mA/180V.
• Semi-dry transfer to PVDF membrane for 2 h at 250 mA/15 V.
• Block the membrane with TBST + 5% non-fat dry milk for 1 h at RT.
• Incubate with primary
• • rabbit anti-RHOT1 antibody (antibodies-online, ABIN405704) diluted 1:1000 in TBST + 3% BSA overnight at 4°C.
  • rabbit anti-Myc1 antibody diluted 1:1000 in TBST + 3% BSA overnight at 4°C.
  • rabbit anti-Cyt c antibody diluted 1:1000 in TBST + 3% BSA overnight at 4°C.
• Wash membrane 3x for 5 min with TBST buffer.
• Incubate with secondar goat anti-rabbit IgG HRP antibody (Sigma, A0545-1M2) diluted 1:2000 in TBST + 5% non-fat dry milk for 1 h at RT.
• Wash membrane 3x for 10 min with TBST buffer.
• Reveal protein bands on a Bio-Rad imaging system using homemade ECL prepared according to PMID 17141265.
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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 RHOT1

Target: RHOT1 (Ras Homolog Gene Family, Member T1 (RHOT1))

Alternative Name: RHOT1

Background: RHOT1 is mitochondrial GTPase involved in mitochondrial trafficking. It is probably involved in control of anterograde transport of mitochondria and their subcellular distribution.
Alias Symbols: ARHT1, FLJ11040, FLJ12633, MIRO-1, MIRO1
Protein Interaction Partner: PCSK9, ERLIN2, PARK2, ILK, UBC, CFTR, PINK1, UBD, IRAK1, AMFR,
Protein Size: 659

Molecular Weight: 75 kDa

Gene ID: 55288

NCBI Accession: NM_001033566, NP_001028738

UniProt: Q8IXI2