1 kb DNA Ladder in 1% agarose gel
• For sizing and quantification of double strand DNA fragments.
• Composed of ten bands as shown on right.
• The 4 kb band with higher concentration is easily distinguishable from the others.
• Premixed with 6X DNA loading buffer for direct gel loading.
1 kb DNA Ladder in 1% agarose gel
• For sizing and quantification of double strand DNA fragments.
• Composed of ten bands as shown on right.
• The 4 kb band with higher concentration is easily distinguishable from the others.
• Premixed with 6X DNA loading buffer for direct gel loading.
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.
Collagen is a fundamental component of the extracellular matrix, and the predominant protein in animals, constituting around 30% of total protein mass. A glycoprotein, it is well known for its triple helical structure. This is formed from three polypeptide α-chains with Gly-X-Y repeating residues (Gly for Glycine, X for proline, and Y for hydroxyproline).
Over 28 types of collagens have been identified, with Type I collagen being the most abundant. It’s prevalent in ligaments, tendons, skin, and bone tissue. Its mature, insoluble form grants it remarkable strength, making it vital for the mobility of organisms. Collagen also has biochemical functions, influencing cell growth, proliferation, and differentiation.
This version of the kit is designed to detect and measure SOLUBLE forms of collagen. Chose the Sircol Insoluble collagen kit if you need to analyse INSOLUBLE collagen.
Collagen, with its diverse properties, finds utility in various industries. It plays a role in medicine for wound healing and has an expanding role in tissue engineering and cell culture for biomedical purposes. It’s gaining popularity in the cosmetic industry for skin rejuvenation and is used in chemical formulations and the food industry as a functional food supplement and additive.
The Sircol 2.0 dye reagent includes Sirius Red, a linear anionic dye with sulfonic acid side chains. This reagent is specially formulated to bind to the Gly-X-Yn helical structure of soluble collagen under assay conditions.
*The improved formulation of Sircol 2.0 dye enables a greater degree of dye-collagen specificity (compared to our previous S1000 assay kit).
Step 1. Prepared samples are placed in the wells of the assay microplate, together with Sircol Dye Reagent. After 30 minutes mixing, any collagen-dye complexes will form as a precipitate. These are collected on the base of the microplate wells by centrifugation.
Step 2. Unbound dye is removed by gentle aspiration, followed by a rinse with Plate Wash Reagent.
Step 3. Following further centrifugation, collagen-bound dye is eluted by incubation with a Dye Release Reagent. Eluted dye is detected ‘in-situ’ by spectrophotometric analysis of the microplate at 556nm.
Step 4. The collagen content of unknown samples can be quantified by comparison against a calibration curve, prepared using the Collagen Reference Standard supplied with the kit.
A list of suggested sample types can be found under the ‘Assay Specification‘ tab.
2 – 200µg/ml or 0.25 – 20µg/ml (using optional Collagen Concentration Protocol)
2 µg/ml
Colorimetric Detection (556nm) (Endpoint), Requires a microplate centrifuge.
96 in total (allows a maximum of 41 samples to be run in duplicate alongside a standard curve).
Soluble* collagens from mammalian**:
In-vivo: Tissues, cartilages and fluids.
In-vitro: Extracellular matrices / Conditioned media from 2D/3D culture environments.
The straightforward sample processing and analysis of Sirco 2.0 make it a good alternative to conventional hydroxyproline analysis.
*Prior salt/acid/acid-pepsin extraction may be necessary to release soluble collagen.
**Sircol 2.0 is primarily designed for use with in-vivo / in-vitro samples of mammalian origin. Collagens originating from other taxonomic groups and kingdoms can also be analysed. See note on p6 of manual for further information.
This kit is designed for research use only. Not for use in diagnostic procedures.
Kit requires access to a microplate centrifuge* (see note below), as well as a spectrophotometer/colorimeter capable of absorbance detection at 556nm.
Specific sample preparation protocols may require customer to provide further reagents, consult assay manual for further information.
*As a minimum, we recommend that the centrifuge can centrifuge a 96-well microplate at 400 x g for 120 minutes. Higher speed centrifuges are recommended (up to a maximum of 2000 x g), allowing a reduction in centrifuge time.
1. Dye Reagent (1x20ml)
2. Collagen Reference Standard (1x5ml, 200µg/ml of soluble Bovine collagen)
3. Plate Wash Reagent (1x28ml)
4. Collagen Concentration Reagent (1x25ml)
5. Neutralisation Reagent (1x8ml)
6. Dye Release Reagent (1x25ml)
7. Assay Microplate (1×96-wells)
8. Microplate Seals (6x)
9. Documentation (QuickStart Guide / Manual / Certificate of Analysis)
NB: Additional reagents may be required for sample preparation prior to assay. Consult manual or contact us for further details. This kit requires the use of a microplate centrifuge, capable of centrifuging a 96-well microplate at 400 x g for 120 minutes. Higher speed centrifuges are recommended (up to a maximum of 2000 x g), allowing a reduction in centrifuge time.
Experience user-friendly detection & measurement of Soluble Collagen with Sircol™ 2.0! Our latest kit simplifies collagen quantification within in-vivo / in-vitro samples. Sircol 2.0 offers enhanced sensitivity and accuracy compared to our previous Sircol kit.
N-(endo-BCN-PEG2-amido-PEG3)-N-bis-(PEG3-Amino-Tri-(Propargyl-PEG2-ethoxymethyl)-methane) is is a multi-functional PEG linker with six terminal propargyl groups and a BCN group. The propargyl groups enables formation of triazole linkage with azide-bearing compounds or biomolecules in copper catalyzed Click Chemistry. The BCN group can react with azide -tagged compound or biomolecules. The hydrophilic PEG spacer increases solubility in aqueous media. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.
N-(endo-BCN-PEG2-amido-PEG3)-N-bis-(PEG3-Amino-Tri-(Propargyl-PEG2-ethoxymethyl)-methane) is is a multi-functional PEG linker with six terminal propargyl groups and a BCN group. The propargyl groups enables formation of triazole linkage with azide-bearing compounds or biomolecules in copper catalyzed Click Chemistry. The BCN group can react with azide -tagged compound or biomolecules. The hydrophilic PEG spacer increases solubility in aqueous media. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.
