DBCO-PEG4-biotin is a click chemistry biotinylation that can enable copper-free click chemistry with azide molecules to form a stable triazole linkage. PEG4 spacer increases aqueou solubility of the molecules conjugated to the biotin compound. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.
DBCO-PEG4-biotin is a click chemistry biotinylation that can enable copper-free click chemistry with azide molecules to form a stable triazole linkage. PEG4 spacer increases aqueou solubility of the molecules conjugated to the biotin compound. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.
ProbeSure Multiplex Master Mix is an enhanced version of ProbeSure Master Mix, formulated to enable users to analyse up to four targets in one reaction well. For example, two bi-allelic SNPs or one reference gene and a further three genes of interest.
Users will require a plate reader capable of reading FAM, HEX, ATTO 550, ATTO 647N and ATTO 633 (the wavelengths of each of these can be found in our ProbeSure Multiplex Master Mix User Guide). ProbeSure Multiplex Master Mix is supplied at 2x concentration for convenience and is supplied with the ATTO 633 normalising dye at either high level (500 nM final concentration), low level (25 nM final concentration) or without ATTO 633.
Apoptosis is an essentially normal physiological process that removes now redundant, cells, particularly during embryonic development and early growth. In adult animals the process removes cells that are irreparable. The apoptotic process is also involved in many major diseases such as cancer, where transformed tumour cells have their apoptotic process disabled, permitting cell cycling to continue unchecked. In contrast some forms of senile dementia may result from excessive apoptotic induction of neural cells.
The apoptotic process in mammalian cells is a rapid event (2‐4 hours). Within this short time span an apparently viable cell can be quietly dismantled, to disappear leaving no visible trace of its former existence.
An apoptosis cascade of activators, effectors and regulators has been identified. This in turn led to a range of apoptosis assays being devised to detect and monitor these events. Some laboratories will employ two distinct assays, one selected to detect early (initiation) apoptotic events, while a second assay will target a later (execution) event. Apoptosis assays, based on methodology, can be classified into four major inter‐linked groups:
[1] DNA fragmentation (electrophoresis and nick end labelling, TUNEL).
[2] Apoptotic proteases (fluorescently labelled antibodies to the caspases).
[3] Flow cytometric analysis (FACS, incorporating other group assays).
[4] Membrane alterations (phosphatidylserine flip).
Biocolor’s APOPercentage assay is based on the latter. Further information can be found under the ‘Mode of Action’ Tab.
The mammalian cell membrane has been described as a semi‐fluid mosaic structure, composed of phospholipids with a diverse group of inserted proteins and some cholesterol. The phospholipids are the major components of the membrane and are arranged in the form of a ‘bi‐layer’; which is asymmetric in composition, structure, and function.
To ensure normal transmembrane functions the phospholipids must be maintained in an asymmetric composition. The process is regulated by ‘flippases’, which catalyse the active transport of aminophospholipids from the outer to inner monolayer. However, in cells undergoing apoptosis, flippase is overwhelmed by the action of another enzyme, termed ‘floppase’ or ‘scramblase’. The net effect is a scrambling of the phospholipid distribution between the inner and outer monolayers.
The APOPercentage assay utilises an intense, pink-coloured dye reagent which is taken up during in-vitro culture by apoptosis-committed cells. This uptake occurs at the stage of Phosphatidylserine transmembrane movement, as produced by the flipflop mechanism. Dye uptake continues until blebbing occurs. No further dye can then enter the now defunct cell and the dye that has accumulated within the cell is not released (unlike necrotic cells which release dye).
Since the dye reagent is excluded or not retained by healthy or necrotic cells it therefore acts as a specific label for apoptotic cells.
Labelled apoptosis cells may then by conveniently analysed by the following methods:
Direct Analysis
The intense pink colour of the labelled cells can be visually assessed using brightfield microscopy. Apoptosis in substrate-adherent cell populations is therefore readily quantified using image analysis techniques. This technique is the most sensitive with the ability of detecting one single apoptotic cell per well.
Colorimetry protocol
Dye that accumulates within apoptotic cells is released into solution via addition of Dye Release Reagent. The concentration of this intracellular dye is then measured at 550nm using a microplate colorimeter/spectrophotometer.
NB: The APOPercentage assay kit does NOT require the use of a Flow Cytometer.
A single cell (via image analysis method)
Colorimetric (550nm) (Endpoint) or Image Analysis based
Sufficient for 4×24 well plates or 6×96 well plates
Adherent mammalian cells (in-vitro)
1. APOPercentage Dye (1x5ml)
2. Dye Release Reagent (1x150ml)
3. Phosphate Buffered Saline (PBS) (1x120ml)
4. 24-well starter plate.
5. Assay kit manual.
The Colorimetric Protocol requires a Microplate Colorimeter / Spectrophotometer.
Additional 96-well plates will be required for use when reading dye absorbance values.
The Direct Detection Protocol Requires an inverted stage microscope with an attached digital camera.
NB: Additional reagents (typically culture medium and suitable apoptosis treatments) may be required for sample preparation prior to assay. Consult manual or contact us for further details.
The APOPercentage™ Apoptosis kit is a dye-based, colorimetric assay for detection and measurement of apoptosis (programmed cell death) during in-vitro cell culture.
Product Description
Kit Storage and term of Validity
Storage term: stored at ≤-20℃,keep away from light, avoid heavy weight and repeated freezing and thawing.
Term of Validity: 14 months
Isothermal nucleic acid Principle Summary
This kit is based on a rapid nucleic acid amplification technology at room temperature and constant temperature:at room temperature and constant temperature(generally 39℃~42℃),reverse transcriptaseuses specific primers and template RNA tosynthesize cDNA strands,and binds the auxiliary protein and single strand with the help of the protein, the recombinase and the primer form a complex; perform a homology search and bind the target homology domain, at this time a D-loop region is formed at the homology positionand strand exchange begins; accompanied by the recombinase from the complex upon dissociation,the polymerase also bindsto the 3′ end of the primer,initiating chainextension.It is suitable for laboratory-level RNA amplification and RNA amplification for other detection purposes.
Isothermal nucleic acid Product Features
1/ High sensitivity and specificity, short reaction time.
2/ The reagent form is freeze-dried, stable and easy to operate.
3/ The reaction can be operated by metal bath and water bath pot without purchasing expensive PCR apparatus.
Technical Parameters:
| Parameters | Details |
|---|---|
| Product Name | RNA Isothermal Amplification Kit Basic |
| Manufacturer | Amp-future |
| Storage Temperature | -20°C |
| Kit Components | Enzymes, Buffers ,Reagents |
| Packaging | 48 Tests/box |
| Detection Limit | 500-1000copies/µL |
| Shipping | ICE |
| Test Time | 5-20mins |
Isothermal nucleic acid Applications
Suitable for RNA isothermal rapid amplification kit(Basic type)
Primer: Require pair of nucleotide primers with the length of 25-35 bp.
RNA basic kit reaction temperature is 39 to 42℃ and time is 5-20 minutes.
Notes
1/ Please avoid nucleic acid contamination and set blank control during reaction due to the high sensitivity of the kit.
2/ Please take out the required quantity of MIRA reaction units for the experiment, and put the rest under storage conditions when performing the experiment.
