Anti-Complex I Immunocapture antibody [18G12BC2] (ab109798)

Overview

  • Product name

    Anti-Complex I Immunocapture antibody [18G12BC2]
  • Description

    Mouse monoclonal [18G12BC2] to Complex I Immunocapture
  • Host species

    Mouse
  • Tested applications

    Suitable for: Flow Cyt, In-Cell ELISA, IP, ICCmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Cow, Human
  • Immunogen

    Tissue, cells or virus. This information is considered to be commercially sensitive.

  • Positive control

    • Cow heart tissue lysate - mitochondrial extract (ab110338) can be used as a positive control in WB. fibroblasts, HL-60 cells, tissue mitochondria
  • General notes

    This antibody clone is manufactured by Abcam.

    Product was previously marketed under the MitoSciences sub-brand.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

    Find protocol for cross-linking antibodies to beads here.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C. Do Not Freeze.
  • Storage buffer

    Preservative: 0.02% Sodium azide
    Constituent: 99% HEPES buffered saline
  • Concentration information loading...
  • Purity

    IgG fraction
  • Purification notes

    Near homogeneity as judged by SDS-PAGE. The antibody was produced in vitro using hybridomas grown in serum-free medium, and then purified by biochemical fractionation.
  • Clonality

    Monoclonal
  • Clone number

    18G12BC2
  • Isotype

    IgG2b
  • Light chain type

    kappa
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab109798 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt Use at an assay dependent concentration.
In-Cell ELISA Use a concentration of 1 µg/ml.
IP Use a concentration of 0.1 - 1 mg/ml.

100 µg mAb can capture at least 25 µg complex I from 1 mg solubilized bovine heart mitochondria.

ICC Use at an assay dependent concentration.

Target

  • Relevance

    Complex I, or NADH ubiquinone oxidoreductase, is a large protein complex of 950,000 Da molecular weight made up by 45 to 46 different subunits. A total of seven of the subunits of the complex are encoded by mitochondrial DNA, while the remainder subunits are nuclear encoded, which are translated in the cytosol and translocated into the organelle for assembly at the inner membrane. The enzyme complex catalyses electron entry from NADH via a flavin (FMN) and several non-heme iron centers. Complex I is sensitive to a wide range of inhibitors, many of which are pesticides or other common environmental toxins, such as rotenone. Complex I dysfunction is a common cause of genetic OXPHOS defects. Altered functioning of this complex is also thought to contribute to several neurological disorders including Parkinson’s disease and schizophrenia. Also, there is evidence of Complex I involvement in diabetes.

Images

  • Complex I was immunopurified from mitochondria isolated from human heart (HHM), cow/bovine heart (BHM), mouse heart (MHM) and mouse brain (MBM).  The lanes were stained with Coomassie Brilliant Blue R. Bands were excised from the gel and proteolytically digested for mass spectrometry analysis. For the immuno-isolation, 50 μg of mAb (18G12BC2 ab109798) was bound to 5 μl of swollen protein G agarose beads according to protocol described here.

  • Immunocytochemistry image of ab109798 stained fibroblasts cells. The cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 minutes). The cells were incubated with the antibody (ab109798, 1 µg/mL) for 2 hours at room temperature or over night at 4°C. The secondary antibody was (green) Alexa Fluor® 4884 goat anti-mouse IgG (H+L) at a 1/1000 dilution for 1 hour. 10% Goat serum was used as the blocking agent for all blocking steps. The target protein locates to the mitochondria.
  • HL-60 cells were stained with 1 µg/mL Complex I antibody ab109798 (blue) or an equal amount of an isotype control antibody (red) and analyzed by flow cytometry.
  • Overlay histogram showing HepG2 cells stained with ab109798 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab109798, 2µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells ) used under the same conditions. Acquisition of >5,000 events was performed.

References

This product has been referenced in:

  • Abuaita BH  et al. Mitochondria-Derived Vesicles Deliver Antimicrobial Reactive Oxygen Species to Control Phagosome-Localized Staphylococcus aureus. Cell Host Microbe 24:625-636.e5 (2018). Read more (PubMed: 30449314) »
  • Zhang Y  et al. Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain. J Biol Chem 292:10239-10249 (2017). Read more (PubMed: 28458255) »
See all 13 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Question
Answer

The image is not of a blot but of a gel stained for total protein. The annotations refer to subunits of mouse brain mitochondria Complex I, identified by mass spec as described in the reference at this link: http://www.mcponline.org/content/4/1/84.full.pdf+html .

Schilling B et al. Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line. Mol Cell Proteomics 4:84-96 (2005).

Read More

Answer

This antibody is an immunocapture antibody which can pull down the whole complex I from a given sample. It works also as a ICC and Flow antibody. It's a weak WB antibody, light up a 8kda subunit of complex I but we have difficulty to identify which subunit it exactly targets on. It has not been tested for its use in BN-PAGE. For BN-PAGE we highly recommend to use ab110412.

Read More

Question
Answer

whole (45 subunit, 900 kDa) active bovine Complex I - this was shown with mass spec.

It is excellent at binding native, active complex I but has proven difficult to identify the exact subunit to which it binds.

Read More

Question
Answer

Thank you for contacting Abcam.

I have attached a paper that looks at the structure of complex 1 and Table 1 has a complete list of the subunits and I believe (but I am no expert in this field) that S1 is also known as NDUFS1.

Please let me know if there is anything else I can help you with.

Read More

Answer

Thank you very much for your phone call.
I have had discussion with my colleague in lab. They have suggested that the antibody was tested only with lysates and cells as presented on the datasheet. No other tested data is available for this product.
I am sorry we are unable to provide further details. The antibody is fully characterized and is guaranteed to for IP, ICC, In-Cell ELISA, Flow Cyt irrespective of protocols used.
I hope this information will be helpful. Should you have any other inquiry please do not hesitate to ask.

Read More

Answer

Thank you for contacting us. I am not sure which publication you are looking at however I have the following publication from Sashi Nadanaciva; http://www.mitosciences.com/PDF/MitoSciences_OXPHOS_Screen_Paper.pdf In this article MS101c is used as a Complex I capture antibody; which helps binding the Complex I to ELISA plate. Now check careful the second paragraph on page 3; "Complex I activity was measured by adding an assay solution containing 25mM KH2PO4 pH 7.2, 5mM MgCl2,1.2% (w/v) bovine serum albumin (BSA), 0.15mM Coenzyme Q1 and 0.26mM NADH. The oxidation of NADH was monitored by measuring its decrease in absorbance at 340 nm in a Spectra Max Plus384 plate reader (Molecular Devices) in kinetic mode at 30 °C for 90min. The rate was linear during this period" Now check the following Kit datasheet https://www.abcam.com/Complex-I-Enzyme-Activity-Microplate-Assay-Kit-ab109721-protocols.html ab109798 alone can’t be used to detect the enzyme activity; it will only bind the complex to the ELISA plate and then you will need further reagents which get reduced by complex 1 for activity measurement. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Thank you for contacting us. I suppose you have a question about ab109798; Anti-Complex I Immunocapture antibody. This antibody detects the presence of Complex I in cell sample; this unfortunately is not suitable for assaying the enzymatic activity. You need to select other products kits e.g. ab109721, ab109720 or other. Rotenone is an Electron Transport Chain inhibitor; it does not affect the biogenesis of mitochondrial complexes so this antibody will not be able to find difference between the Rotenone treated and Rotenone untreated cells.   For more information please click the following links http://www.mitosciences.com/mitobiogenesis-in-cell-elisa-kit-colorimetric.html http://www.ruf.rice.edu/~bioslabs/studies/mitochondria/mitopoisons.html I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Thank you for contacting us. Please click these links to find the suitable antibodies. Type the MS code in Abcam search box to get the Abcam catalogue number. Complex I http://www.mitosciences.com/complex_i.html Complex IV http://www.mitosciences.com/complex_iv.html Complex III http://www.mitosciences.com/complex_iii.html Complex II http://www.mitosciences.com/complex_ii.html We also have Immuno capture kits available https://www.abcam.com/index.html?pageconfig=resource&rid=13917 I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up