Cupid Peptides arrive in a vacuum-sealed format in packs of 1 UNIT. They are designed to be used straight out of the packaging with minimal fuss. They do not contain TFA or Acetate so do not require lengthy clean-up steps. The only step before adding to cultured cells involves making certain the peptide has dissolved thoroughly in water.
Cupid Peptides are dried down, stored and shipped under vacuum. Inside the Vacuum-sealed pack is a plastic vessel containing the Cupid peptide which has a perforation in the lid to maintain the vacuum in the tube. This lid should be replaced by a non-perforated lid (supplied) when the package is first opened. Unopened peptide packages can be stored at 4 degrees for at least 1 year. After reconstitution in water, the peptide becomes susceptible to bio-degradation. Any unused stock solution should either be frozen and used within a month or alternatively dried down and kept in an airless state where it can be stored for at least the remainder of the shelf life duration. It is not recommended to store peptide after it has been diluted in media or buffer.
Cupid peptides are supplied in quantities of 1 Unit, where 1 Unit is 10 nanomoles of peptide. When fully dissolved in 100 microlitres of distilled water, 1 Unit will create a 100 micromolar (x10) Stock Solution and is sufficient to dose 1 mL of cell culture at the maximum recommended final concentration of 10 micromolar. We determine the concentration of all Cupid peptides using a commercial bicinchoninic acid (BCA) protein assay, using Bovine Serum Albumin (BSA) to generate a standard curve at an absorbance of 562 nanometers vs a water blank. Based on this curve, the peptide molarity is then calculated using the theoretical molecular weight of each Cupid Peptide (determined using protein programs available online such as: http://web.expasy.org/protparam/). The concentration of Stock Solutions may be tested in the same way.
Steps for solubility
* Upon first opening the vacuum package, replace the perforated lid that is on the cupid peptide tube with the non-perforated lid provided.
* To make a 100 micromolar (x10) Stock Solution add 100 microlitres of distilled water to 1 Unit of peptide.
* Leave the peptide to dissolve for 15-30 minutes at room temperature with occasional agitation. Please be patient. A small amount of base might speed the process (start with 0.5 millimolar NaOH). Ensure any base added can be buffered by the final media used.
* Once the solution is clear, dilute the peptide to the desired concentration in cell media. We recommend a maximum dose for experiments of 10 micromolar. If sterile conditions are required, filter the (peptide + media) solution through a sterile 0.22 micron syringe filter (PES is recommended for this purpose). Note: some loss of volume can be expected.
* Replace the media in the cell culture with the (peptide + media) solution.
* The peptide will permeate the cells and build up in them to equilibrium in 45-90 minutes. Biological effects may be observed before this.
Guarantees of Cupid Peptides
We provide Cupid peptides to the customer with 3 essential guarantees that we know are important for their use with cultures of living cells.
1. Solubility* The peptides are soluble in water to at least 200 micromolar concentration (x20 recommended maximum dose).
2. Purity** The purity of the peptide is in excess of 80% unless otherwise stated.
Note: Any protein impurities do not possess the Cupid cell-permeating sequence and do not have the capability to permeate living cells. The Cupid Control Peptide (Cat: Cupid-Control) is available to control against any bioactivity of any protein impurities
3. Cell Permeation*** All Cupid products are tested for their capability to penetrate living cells at the maximum recommended dose within 1 hour of addition. Under these conditions we have found fluorescein-labelled Cupid peptides to be effective in permeating into a wide variety of living cells including Human Kidney, Human Fibroblast, Chicken neurones and Amoebae.
*The stock solution remains soluble when diluted to 10 micromolar with common media formulations or 20 mM phosphate buffer or PBS (137 mM NaCl) at pH 7-8
**The identity of the peptide is confirmed by running peptide samples on SDS-PAGE gels followed by (a) Protein staining to confirm the major peptide band agrees with the molecular weight calculated for the Cupid peptide (b) Fluorescent staining of a Cupid-specific motif identifies this major peptide band as being the Cupid-linked product.
***This is performed by first labelling each peptide with fluorescein and repurifying it. Labelled peptide is then incubated with living cells at the maximum recommended dose for use (10 micromolar) for 60 minutes before exchanging the media and washing the cells. Cells are finally prepared for imaging under a fluorescent microscope (Excitation wavelength of 494nm and an Emission wavelength of 518nm). Fluorescent images of treated cells are taken at a setting where the background (autofluorescence) of the untreated cells is at the threshold of detection. It is noted that fluorescence is observed evenly diffused throughout all cells. It is not seen lining the cell membranes and rarely seen in any local concentration except where cells have clumped to give a relative additive effect.