Frequently Asked Questions
Lipid Strips and Arrays
PIP Mass Assays
Echelon Biosciences currently carries 5 PIPn Mass ELISA kits that can measure the relative levels of 5 different PIPn. Each kit uses a lipid extraction protocol designed for the extraction of phosphoinositide (PIPn) which are then analyzed in a competitive ELISA. PIP Mass FAQs are below. For more detail, please see our full PIP Mass FAQ document.
The amount of cells necessary for quantification should be determined for each cell type and PIPn. Suggested starting points can be found in the product specific technical data sheet. If larger or smaller amounts of cells are required the extraction volumes will need to be proportionately adjusted.
Echelon Biosciences currently carries a variety of assays to measure activity of Class I and Class III PI3-kinases. See our PI3K Assay Comparison Guide.
Hyaluronic Acid Assays
Echelon Biosciences carries a variety of assays for detecion of hyaluronic acid (HA). See our HA Assay Comparison Chart.
Echelon Biosciences carries antibodies directed at specific lipids. These may be generally used in the same manner as protein antibodies but special considerations may apply depending on the assay and antibody.
Echelon’s Shuttle PIPTM kits are designed for researchers interested in visualizing and localizing intracellular phosphoinositides. The kits contain labeled and unlabeled carrier proteins for the intracellular delivery of fluorescent and nonfluorescent phosphoinositides. See the Shuttle PIP FAQ for additional details.
Common questions about handling peptides can be found below.
The stability is dependent on the sequence since some amino acids are less stable than others. Cys, Met, & Trp are prone to oxidation. Many peptides are stable for years when stored at -20 oC, though some can have shorter shelf lives.
Where known we have a suggested solvent on the CofA. Peptide solubility is related to the amino acid sequence and conformation. While the solubility cannot always be predicted from the sequence, some assumptions can be made. Since peptides purified with acid (generally trifluoroacetic acid), the presence of basic amino acids (Arg, Lys, His) will increase the solubility in water since those groups will be protonated. The water solubility of peptides containing acidic amino acids (Asp & Glu), can be increased by adding dilute ammonium hydroxide forming the ammonium salt. Peptides with large numbers of non-polar residues (Ala, Val, Ile, Leu, Trp, Tyr, Val) likely will require the addition of an organic solvent like DMSO, DMF, or acetonitrile to the aqueous suspension to help the peptide into solution. Bath sonication for a few minutes will break up aggregates.
pNA substrates: λmax 405 nm
AFC substrates: λex = 395-400 nm, λem = 495-505 nm
AMC substrates: λex = 360-380 nm, λem = 440-460 nm
MCA substrates: λex = 325 nm, λem = 392 nm
EDANS Substrates: λex = 365 nm, λem = 490 nm
4MβNA substrates: λex = 335-350 nm, λem = 410-440 nm
Trp/DNP substrates: λex = 280 nm, λem = 300-350 nm
Abz substrates: λex = 320 nm, λem = 420 nm
The gross weight of a peptide includes the mass of the peptide, counter-ions, and residual water molecules. Depending on the formulation, the counterions are typically trifluoroacetate, acetate or chloride.
Peptide content is the percentage of free peptide in the gross weight relative to counter ions and residual water. Peptides with a high proportion of basic residues (Lys, His, Arg) will have lower peptide content due to increased amount of counterions.
The net weight is calculated by multiplying the gross weight x % peptide content
e.g. When the peptide content = 79%: 1 mg * 0.79 = 0.79 mg net weight