RHOT1 antibody (N-Term)

Catalog No. ABIN635090

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 and Rat.

Quick Overview for RHOT1 antibody (N-Term) (ABIN635090)

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

Reactivity: Human, Mouse, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This RHOT1 antibody is un-conjugated

Application: Western Blotting (WB)

Product Details anti-RHOT1 Antibody

Binding Specificity: N-Term

Specificity: RHOT1 antibody was raised against the N terminal of RHOT1

Purification: Affinity purified

Immunogen: RHOT1 antibody was raised using the N terminal of RHOT1 corresponding to a region with amino acids EMKPACIKALTRIFKISDQDNDGTLNDAELNFFQRICFNTPLAPQALEDV

Application Details

Application Notes: WB: 1 µg/mL
Optimal conditions should be determined by the investigator.

Comment:

RHOT1 Blocking Peptide, (ABIN937971), is also available for use as a blocking control in assays to test for specificity of this RHOT1 antibody

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100007 (Western Blotting)

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

No.: #100007

Date: 09/12/2015

Antigen: Ras Homolog Gene Family, Member T1 (RHOT1) (N-Term)

Method validated: Western Blotting

Positive Control: 100ng Recombinant Miro1; 30ug Mitochondria isolated from Miro1 WT MEFs and Miro1 KO MEFs expressing Myc-Miro1; in house purification and isolation Isolated mitochondria from immortalized Miro1 WT MEFs rMiro1, mtMiro1 wt MEFs, mtMiro1 KO MEFs plus Venus-Miro1

Negative Control: 100ng Recombinant Miro2, 30ug Mitochondria isolated from Miro1 KO MEFs and Miro1 KO MEFs expressing Myc-Miro2; in house purification and isolation

Notes: ABIN635090 is specific for Miro1 without cross-reaction with Miro2.

Validation Images

Western blot customer validation ABIN635090. Refer to the protocol section for more information.

Full Methods

Primary Antibody: ABIN635090

Secondary Antibody: Goat-anti-rabbit IgG HRP conjugated (Sigma A0545-1M2 - 057K4802) 1:2000 in TBS + 0.05% Tween-20 + 5% non-fat dry milk

Full Protocol:

• Transfection of immortalized Miro1-/- MEFs with Myc-Miro1-V1 or Myc-Miro2-V1 (Published in Nguyen et al., 2014 (PNAS, 10.1073/pnas.1402449111))
• Isolation of mitochondria from immortalized Miro1+/+, Miro1-/-, Miro1-/- + Myc-Miro1 and Miro1-/- + Myc-Miro2 MEFs
• Determine protein concentration and snap freeze mitochondria in liquid nitrogen

  Day 1

• Thaw 30ug mitochondria per lane on ice
• Thaw 100ng recombinant Miro1 and Miro2 per lane on ice (in house purification)
• Add 2x Laemmli buffer + 2% BME to samples and denature for 5 min at 95C
• Load 30ug/lane mitochondria and 100ng/lane recombinant protein onto 12.5 % SDS-PAGE
• Fermentas PageRuler Plus Prestained (2ul) - Lane 7 / 14 / 21
• Recombinant Miro1- Lane 1 / 8 / 15
• Recombinant Miro2- Lane 2 / 9 / 16
• Miro1+/+ mitochondria- Lane 3 / 10 / 17
• Miro1-/- mitochondria- Lane 4 / 11 / 18 / 22
• Miro1-/- + Myc-Miro1-V1 mitochondria- Lane 5 / 12 / 19 / 23
• Miro1-/- + Myc-Miro2-V1 mitochondria- Lane 6 / 13 / 20 / 24
• Run gel 40mA 180V for 3h
• Semi-dry transfer to PVDF 250mA 15V for 2h
• Block with TBS + 3% BSA for 1h at RT

1st AB over night at 4C rocking

Lanes	Antibody	Host	Dilution	In
1-6	Anti-Miro1 Other company	-	-	-
8-13	Anti-Miro1 ABIN634527v	rabbit	1:1000	TBST + 3% BSA
15-20	Anti-Miro1 ABIN635090	rabbit	1:1000	TBST + 3% BSA
22-24	Anti-Myc	-	-	-
All lanes	Anti-Cyt c	-	-	-
Anti-PRX3	-	-	-	-

Day 2

• 3x washing with TBS + 0.05% Tween-20 for 5'
• 2nd AB for 1h at RT
• Goat-anti-rabbit IgG HRP (Sigma A0545-1M2 - 057K4802)
• 1:2000 in TBS + 0.05% Tween-20 + 5% non-fat dry milk3x washing with TBS for 10'
• Detection with homemade ECL prepared according to Haan and Behrmann, 2007 (J Immunol Methods, 10.1016/j.jim.2006.07.027) on a Bio-Rad imaging system

Handling

Format: Lyophilized

Reconstitution: Lyophilized powder. Add distilled water for a 1 mg/mL concentration of RHOT1 antibody in PBS

Concentration: Lot specific

Buffer: PBS

Handling Advice: Avoid repeated freeze/thaw cycles.
Dilute only prior to immediate use.

Storage: 4 °C/-20 °C

Storage Comment: Store at 2-8 °C for short periods. For longer periods of storage, store at -20 °C.

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.

Molecular Weight: 75 kDa (MW of target protein)