RHOT1 antibody (N-Term)

Catalog No. ABIN635090

Product Details anti-RHOT1 Antibody

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

Binding Specificity: N-Term

Reactivity: Human, Mouse, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This RHOT1 antibody is un-conjugated

Application: Western Blotting (WB)

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, catalog no. 33R-2585, 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 (RHOT1 Products)

Synonyms: rhot1 antibody, zgc:55581 antibody, zgc:77063 antibody, wu:fd14e11 antibody, wu:fi14a05 antibody, arht2 antibody, miro-2 antibody, rasl antibody, ARHT1 antibody, MIRO-1 antibody, MIRO1 antibody, 2210403N23Rik antibody, AA415293 antibody, AF244542 antibody, AI834919 antibody, Arht1 antibody, C430039G08Rik antibody, Miro1 antibody, miro1 antibody, si:dkeyp-97g3.6 antibody, ras homolog family member T1a antibody, ras homolog family member T2 antibody, ras homolog family member T1 antibody, ras homolog family member T1 L homeolog antibody, rhot1a antibody, rhot2 antibody, RHOT1 antibody, rhot1 antibody, rhot1.L antibody, Rhot1 antibody, rhot1b antibody

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)