Tech Notes

Glycobiology Tech Notes


TNGP100 GlykoPrep Guidebook
TNGP101 UPLC Rapid Methods
TNFC300 FACE N-Linked Glycan Sequencing
TNGL100 Principles of Glycobiology
TNGL101 Biosynthesis of N- and O- Glycans
TNGL102 GPI Membrane Anchors
TNGL103 Unusual Modifications
TNGL104 Biological Roles of Oligosaccharides
TNGL105 Pharmacological Effects of Glycosylation
TNGL106 The Role of Glycosylation in Disease
TNGL107 Selectins and Glycosylation in Inflammation
TNGL108 Plant Lectins
TNGL109 Animal and Bacterial Lectins
TNGL110 Lectins as Indicators of Disease


Phycobiliprotein Tech Notes



Phycobiliprotein Overview

Including nomenclature, structure, properties, and detailed descriptions.


Techniques for Optimizing the Sensitivity of Fret Assays

Homogeneous FRET assays have become popular for the detection of molecular interactions, driven both by the inherent robustness of fluorescence assays and by the logistic simplicity of their implementation. Perceived limits to their sensitivity, however, have mitigated against their use with lower affinity molecular interactions. Through a systematic reexamination of FRET assay design and signal detection, opportunities for significantly enhanced assay sensitivity can be identified.


Multiple-Lot Comparison of PhycoLink PJ25S Streptavidin-Allophycocyanin conjugates in a Performance Assay

ProZyme's performance testing of streptavidin-APC demonstrates the historical consistency of the product and assures the consistency of new lots.


Precision of FRET Assays: S/N vs. S/B

The sensitivity of an assay is indicated by its signal-to-noise ratio (S/N). S/N should not be confused with the signal-to-background ratio (S/B), which can provide misleading indications when improperly interpreted as equivalent to S/N.


Measuring the Precision of FRET Assays: S/N and Z'

S/N and Z' are useful indices of assay precision for FRET assays, and incorporate the same assay response parameters. The choice between them should be based on the needs of the investigator: Z' is particularly sensitive in discriminating between assays with poor precision; S/N provides clearer distinctions between higher precision assays.


Background Correction and Spectral Overlap Compensation in FRET Assays

When proximity between two fluorescent molecules leads to FRET, the total fluorescence emission spectrum of the mixture is different from the spectrum of the same molecules mixed randomly in solution. The spectral differences reflect changes in the magnitudes of the donor and acceptor emission spectra, added together and superimposed on background fluorescence from various sources. These components of the complex emission spectra are identified and discussed to illustrate the principals behind the various methods of calculating FRET results (TNPJ100.04 FRET Calculations).


FRET Calculations

The calculation of FRET results requires both correction for blanks and compensation for spectral overlap between channels. Moreover, the final results of FRET assays may be expressed in several different ways, either in terms of FRET counts or as ratios. Equations are provided for these various output parameters.


Dissecting FRET Data: Quench-FRET Analysis

Quench-FRET analysis goes beyond standard FRET parameters (such as A/B ratio and Net FRET) by examining donor Quench, FRET and their ratio (Q/F). It is useful for detecting false positives and other artifacts produced by interference from absorbent/fluorescent sample compounds. Appropriate for both TR-FRET and PB-FRET assays, it is particularly suited to the latter because of the strong donor Quench and low noise in PB-FRET assays.



Phycobiliprotein-FRET (PB-FRET) can achieve signal-to-noise ratios significantly higher than those achieved with time-resolved FRET (TR-FRET).


Selection of Donor and Acceptor Reagents in a TR-FRET Assay

TR-FRET assays in which the more costly lanthanide fluor, rather than the less expensive phycobiliprotein fluor, is conjugated to streptavidin provide similar performance at a lower overall cost.


Microplate Color Comparison in a TRF Assay

Time-resolved FRET (TRF) assays were originally developed to overcome problems with sample background autofluorescence in proximity assays. Through the use of long lifetime fluorescence donors, detection is delayed until fluorescence from short-lived sources subsides, thereby eliminating most background.


Microplate Color Comparison in a TRF Assay

PB-FRET results that may be compromised due to candidate absorbance or fluorescence are readily identified by Quench-FRET analysis. The cost of the additional controls is minimal compared to the improved discrimination capability.


Self-quenching in FRET Assays

At high reagent concentrations, fluorescent reagents can reabsorb their own emitted fluorescence, leading to unexpected nonlinearity in reagent concentration effects on fluorescence.


PB-FRET™: Ilumination and Detection Windows for Filter-based Instruments

By examining excitation and emission spectra for donor and acceptor fluors, optimum detection windows for FRET assays can be established.


Suitability of Fluorescent Molecules in Fret Assays

The cyanine dye Cy5 has similar spectral characteristics to APC but is a weaker fluorescence acceptor. APC-streptavidin gave six times the FRET counts and twice the signal:noise as Cy5-streptavidin in a tyrosine kinase TR-FRET assay.


PhycoLink® Conjugate Evaluations

Describes techniques to evaluate PhycoLink conjugates via absorbance, affinity chromatography, and HPLC/gel filtration. Methods for calculating concentrations, molarities and molecular weights of fluorescent molecules and conjugates.


Conjugate Brightness


How to Make the Best Darn Conjugates (~10MB)

Researchers increasingly conjugate their own antibodies because they want direct conjugates; have only limited quantities to work with; can't find the right color on the desired marker; want to reserve the brightest tags for their dimmest antigens; or are driven by the need for more cost-effective reagents. Others may just want to do it themselves or understand the basic principles. We've got the answers for all of you. Please join us for a tutorial on conjugating PE, APC, PerCP and other phycoproteins (with subsequent conjugate purification) using ProZyme's fast and easy kits. We will also focus on those factors that make conjugates bright, reducing the scale (50 ug or less), evaluating conjugates for consistency lot-to-lot, scaling up and troubleshooting. Get the benefit of years of experience in one short hour from the people who know phycobiliproteins.


How to Make the Best Darn Conjugates - medium resolution (~2.5MB)


Alternative Conjugation Protocols

Protocols for Iminothiolane and SPDP conjugations when the standard protocol doesn't produce an acceptable conjugate.