Complex I Enzyme Activity Dipstick Assay Kit (ab109720)

Overview

• Product nameComplex I Enzyme Activity Dipstick Assay KitSee all Complex I kits ...
• Sample type
  Cell culture supernatant, Cell culture extracts, Tissue
• Assay typeEnzyme activity
• Species reactivity
  Reacts with: Mouse, Rat, Cow, Human
  
• Product overview

  Contains 30 or 90 dipsticks and necessary components to quantify the activity of the Complex I enzyme complex in human, bovine, rat and mouse samples. The kit includes sufficient materials to generate a standard curve and evaluate several unknown samples.
  

  In this assay the specificity of anti-Complex I monoclonal antibodies (mAbs) is combined with the well-characterized Complex I in-gel activity assay that is not rotenone sensitive. First, Complex I is immunocaptured (i.e. immuno-precipitated in active form) on the dipstick. Second, the dipstick is immersed in Complex I activity buffer solution containing NADH as a substrate and nitrotetrazolium blue (NBT) as the electron acceptor. Immunocaptured Complex I oxidizes NADH and the resulting H+ reduces NBT to form a blue-purple precipitate at the Complex I antibody line on the dipstick. The signal intensity of this precipitate corresponds to the level of Complex I enzyme activity in the sample. Combined with dipstick assay kit for measuring Complex I quantity (ab109722/MS131 for human sample; ab109875/MS133 for rodent samples), it is possible to determine the relative specific activity of immunocaptured Complex I. The signal intensity is best measured by a dipstick reader or may be analyzed by a standard imaging system.

• Notes

  Store dipsticks at room temperature in their provided container and out of direct sunlight. High humidity conditions should be avoided.

  Store Buffer A, B, and C at 4°C or at -20°C for long term storage.

  Store Tubes 1 and 2 at -80°C; they can also be aliquoted upon receipt to prevent freeze/thaw cycles.

  Tube 3 can be stored at room temperature.

• Tested applicationsFunctional Studies more details

Properties

• Storage instructionsPlease refer to protocols.

• Components	90 tests	30 tests
  96-well microplate	6 units	2 units
  Buffer B (Blocking solution)	1 x 6ml	1 x 2ml
  Buffer C (Wash buffer)	1 x 3ml	1 x 1ml
  Dipsticks	1 x 90 units	1 x 30 units
  Extraction Buffer	1 x 45ml	1 x 15ml
  Tube 1 (NBT 50X stock)	1 x 600µl	1 x 200µl
  Tube 2 (NADH 100X stock)	1 x 300µl	1 x 100µl
  Tube 3 (Buffer for activity assay)	1 x 30ml	1 x 10ml

• Research Areas
  • Metabolism
  • Pathways and Processes
  • Mitochondrial Metabolism
  • Mitochondrial markers

  • Kits/ Lysates/ Other
  • Kits
  • Cell Metabolism Kits
  • Other Metabolism Assay

  • Signal Transduction
  • Metabolism
  • Mitochondrial

Applications

Complex I Enzyme Activity Dipstick Assay Kit images

• 

  - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)
  Figure 1. An example using ab109720 to measure Complex I activity in human fibroblast samples. Developed dipsticks from a 1:2 dilution series using a positive control sample and the associated standard curve. Starting material was 30 µg of fibroblast protein extract.
• 

  - Complex I Enzyme Activity Dipstick Assay Kit (ab109720)
  Figure 2. An example using ab109720 to measure Complex I activity in human fibroblast samples. Based on the standard curve, 15 µg of protein extract were loaded onto a dipstick for each sample. The figure shows four developed dipsticks, a control sample (1) and three unknowns (2-4). The analysis of the signal intensity and interpolation from the standard curve showed that the unknown samples have between 14-61% of normal Complex I activity levels.
• 

  Functional Studies - Complex I Enzyme Activity Dipstick Assay Kit - 90 tests (ab109720)
  Abcam's enzyme activity assays apply a novel approach, whereby target enzymes are first immunocaptured from tissue or cell samples before subsequent functional analysis. Dipstick ELISA Kits extend this concept by utilizing the well-established lateral flow concept, wherein capture antibodies are striped onto nitrocellulose membrane and a wicking pad draws the sample through the antibody bands. All of our ELISA kits utilize highly validated monoclonal antibodies and proprietary buffers, which are able to capture even very large enzyme complexes in their fully-intact, functionally-active states.

Protocols

Protocol Booklet