Complex I Enzyme Activity Dipstick Assay Kit (ab109720)
- Product nameComplex I Enzyme Activity Dipstick Assay KitSee all Complex I kits ...
- Sample typeCell culture supernatant, Cell culture extracts, Tissue
- Assay typeEnzyme activity
- Species reactivityReacts 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.
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
- 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
- NADH dehydrogenase
Our Abpromise guarantee covers the use of ab109720 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Complex I Enzyme Activity Dipstick Assay Kit images
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.
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.
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.
References for Complex I Enzyme Activity Dipstick Assay Kit (ab109720)
This product has been referenced in:
- Bull VH et al. Sorafenib-induced mitochondrial complex I inactivation and cell death in human neuroblastoma cells. J Proteome Res 11:1609-20 (2012). Read more (PubMed: 22268697) »
- Valentin-Vega YA et al. Mitochondrial dysfunction in ataxia telangiectasia. Blood : (2011). Read more (PubMed: 22144182) »