Frequently Asked Questions

Lipid Strips
PIP Mass Assays
PI3K Assays
HA Assays
Lipid Antibodies
Liposome Stability​
Shuttle PIPs
Peptide Handling

Lipid Strips and Arrays

Echelon Biosciences’ Lipid Strips and Arrays are simple protein-lipid overlay assays used to screen for selectivity and relative affinity between a protein of interest and common cellular lipids. Our recommended protocol for these products can be found here. For additional troubleshooting, please see our full Strips and Arrays FAQ document.
We do not recommend the use of cell lysates with these products as it contributes to high background and can obscure positive signals due to non-specific binding of other cellular components or proteins.
A complete absence of signal (white membrane) is typically due to loss of activity of the binding protein or high detergent concentrations in the assay buffer. In either case, it is recommended to run a positive control for the lipid of interest in order to determine the issue. If aberrant binding is observed this may also be due to harsh detergent conditions or over development of the membrane after application of the detection reagent. Additional details can be found above in our full FAQ document.
All strips and arrays have been internally validated using HRP-conjugated secondary antibodies and can be used with precipitating reagents or chemiluminescent solutions. Use of fluorescently conjugated secondary antibodies is also possible with these products but we encourage the user to optimize the antibody concentration in order to decrease background signal.
High background is most likely attributable to insufficient washing or excess binding protein used in the initial incubation step. Background signal may also be reduced by adjusting the composition of the blocking buffer.

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.

The PIPn extraction protocols have been verified using the cells lines: NIH-3T3, HL-60, MDA-MB-231, and MDA-MB-468. Adherent Cell Lines, NIH-3T3 mouse fibroblasts, MDA-MB-231 human breast cancer, and MDA-MB-468 PTEN deficient human breast cancer were used to validate the assays.
Normalize cells by counting or running a cellular protein assay. Adherent cells can be counted before stimulation or before the cell extraction procedure. Non-adherent cells can be counted before stimulation or before the cell extraction procedure. After stimulation, spin cells down, decant the media and add ice cold 0.5 M TCA and proceed with the cell extraction protocol. Count or perform protein assay on a separate flask with the same cell dilution as the flasks to be used for cell extraction. Counting cells, either by haemocytometer or protein assay, is not recommended after the 0.5 M TCA is added. Do not trypsinize cells before adding 0.5 M TCA; it can alter stimulation time and phosphoinositide levels. The optimal cell number will vary depending on the PIPn being assayed.
Yes, we generally recommend a serial extraction protocol for removal of proteins, neutral lipid, and acidic lipids. The full extraction protocol is available in the 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.

In general the extraction protocol described in the kit will work for tissue as well. The tissue should be homogenized using a detergent buffer to remove tissue debris and insoluble material. The resulting lysate supernatant is then used for the lipid extraction.

PI3K Assays

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.

The choice of assay mainly depends on three factors: the class of PI3K enzyme, the sensitivity, and the sample type. A full comparison guide for our available PI3K Assays can be found here.
In general, analysis of PI3K activity requires a purified enzyme or immunoprecipitated sample. However, our PI3-Kinase Activity ELISA: Pico (cat # K-1000s) can be used with cell lysates and includes a support protocol for this sample type.
No, all of our available PI3K assays are free of radioactivity and are based on absorbance or fluorescence measurements.

Hyaluronic Acid Assays

Echelon Biosciences carries a variety of assays for detecion of hyaluronic acid (HA). See our HA Assay Comparison Chart.

The choice of assay will mainly depend on the sensitivity that is required and the sample type. Please see our assay comparison guide for a full listing of specific assay details.
Yes, except for the HA AlphaScreen, the assays are generally compatible with cell supernatants and serum samples.

Lipid Antibodies

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.

Our lipid antibodies are generated in a manner largely similar to that used for protein antibodies. Synthetic lipids are conjugated to a carrier molecule and the conjugates are then used to generate an immune response in a host animal. The cells generated in the immune response are then used to generate hybridomas which then undergo selection and screening to ensure monoclonality and specificity.
Validated assays will vary by antibody. Internally validated assays can be found under the ‘Applications’ section of the product page or Technical Data Sheet. Assays that have been published in scientific journals but not independently verified by Echelon Biosciences can be found in the reference list for a given antibody.
Yes, general techniques for immunocytochemistry and immunohistochemistry can be applied with lipid antibodies. However, we encourage users to first consider how their immunostaining protocol may affect detection of their lipid of interest. A set of recommended protocols and other considerations can be found here.

Liposome Stability

Echelon Biosciences carries a variety of lipids that can be used to generate liposomes in vitro. Addtional details are available in our Liposome FAQ. A protocol for generating liposomes can be found here.

Liposomes reconstituted in aqueous buffers can be stored at 4C for approximately one week. The pH of the buffer should be kept close to 7. Freeze-thawing of liposomes in aqueous solution should be avoided as this can lead to fracturing and changes in size distribution.
Hydrolytic degradation is a general problem with lipid products. This hydrolysis is mainly dependent on pH, temperature, buffer species, ionic strength, acyl chain length and headgroup, and the state of aggregation. Unsaturated lipids will also oxidize more readily than saturated lipids so lipids that are more highly saturated will have greater stability.

Shuttle PIPs

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.

We have successfully delivered fluorescent phosphoinositides into NIH-3T3, 3T3-L1,Primary cardiac fibroblasts (Rat and Mouse), HeLa, MDCK, HL-60, BMMC (bone marrow derived mast cells), A. thaliana root-tip cells, E. coli, and the protist, C. parvum. Other cell types will need to be tested by the user.
For bisphosphorylated PIPs or PIP3, we suggest trying Histone H1 (Carrier 2) first. If you are trying to deliver monophosphorylated PIPs, Carrier 3 is recommended.

Peptide Handling

Common questions about handling peptides can be found below.

Peptides can be hygroscopic and should be handled rapidly to minimize exposure to moisture, especially in humid environments. Vials from the freezer should be allowed to warm to room temperature before opening.
Lyophilized peptides should be stored in a freezer at -20 oC. Peptides labeled with fluorophores (AMC, AFC, FAM, TAMRA, etc) should be protected from intense light to avoid photobleaching.

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.

 

Peptides in solution are less stable than dried lyophiliates and are best stored at -20oC or below. Multiple freeze-thaw cycles can affect the stability so aliquoting your solution into single-use vials will minimize degradation. For aqueous solutions, pH 5-7 is best. When using organic solvent (e.g. DMSO) dry solvent is preferred to minimize hydrolysis.
Beta Amyloid (1-42) [Ab (1-42)] aggregates readily forming insoluble fibrils. Dissolve 1 mg in 70-80 uL 1% NH4OH then dilute to 1 mg/mL with 1X pH 7.4 PBS. Do not store in 1% NH4OH, dilute immediately with pH 7.4 PBS. Hexafluoroisopropanol (HFIP) can be used to prepare monomeric Ab(1-42). Dissolve 1 mg in HFIP then cap the vial and allow to stand for 30 minutes. Remove the cap and allow the solvent to slowly evaporate (in a fume hood). Dry under vacuum for an additional 2 hours to remove residual solvent leaving a film of monomeric peptide which can be dissolved in pH 7.4 buffer. Alternately, HFIP-treated is available in our catalog (Cat# 641-00).

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

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