Europium Labeling Kit Catalog Number KA2096 100ug *1 Version: 03 Intended for research use only www.abnova.com
Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle of the Assay... 3 General Information... 5 Materials Supplied... 5 Storage Instruction... 5 Materials Required but Not Supplied... 5 Precautions for Use... 5 Protocol... 7 Reagent Preparation... 7 Assay Procedure... 7 KA2096 2 / 8
Introduction Intended Use The Europium Labeling Kit is intended for labeling of molecules with europium through an isothiocyanate reaction to be used in the construction of FRET, QRET & Solid Phase Separation assay formats. Background The Eu-labeling reagent is termed is various applications as the W1024 chelate. The chelate is technically a fluorescent stable chelate. However the europium metal can be disassociated from this chelate into a new highly fluorescent chelate through the use of Enhancement Solution. The W1024 chelate is activated with an isothiocyanate group that reacts with free amino groups on molecules to form a covalent thiourea bond. The kit can be used to label up to 1 mg of IgG to yield 5 10 Eu/IgG. The fluorescence is measured in a time resolved fluorometer. Enhancement Solution The solution dissociate Eu 3+ from solid phase bound Eu-labelled antibodies during a time period of 30 minutes for W1024 & 2-3 minutes for N1 chelates to form a homogeneous and highly fluorescent Eu (2-NTA) 3 (TOPO) 2-3 micellar chelate solution. The solution allows highly sensitive Eu 3+ measurements to be made when using the time-resolved fluorometer. Principle of the Assay Labelling of proteins and other large (>5000 MW) molecules Condition for labelling The conditions depend on the nature of the protein to be labeled. The proteins to be labeled must be dissolved in buffers not containing any amines. Also azides interfere with the reaction. Labeling yield If the labeling protein is to be used in immunoassay applications, the target labeling yield is about 5-15 Eu/protein (IgG). Higher levels may cause protein aggregation & increased non-specific background, as well as decrease storage stability. When labeling antibodies and proteins to be used in immunoassays, the target is to reach at least 5-16 Eu atoms / molecule, but not more than 20 Eu atoms / molecule. Above this figure proteins tend to aggregate and cause higher backgrounds, especially after storage. The labeling yields need to be optimized for each specific biomolecule and assay format. This is especially true of antibodies and other sensitive proteins. The reactivity under labeling is related to the number of free amino groups (i.e. the pi of the protein and its concentration). Lysine-rich proteins are KA2096 3 / 8
more reactive with isothiocyanate than proteins with acidic pi. The kinetics of the conjugation very much depends on the ph and temperature of the reaction. When high yields are needed, the labeling could be conducted at 4 C for 1-3 days or at RT over night. Higher temperature may be used with stable proteins with a shorter incubation time. The labeling kit contains reagent suitable for labeling of up to 1 mg of IgG type of proteins. From protein point of view labeling yield is not dependent on protein amount but reagent concentration, unless targeting excessive quantities of proteins. Basically, at a given condition and reagent concentration, 0.1 mg of protein are labeled at a same level as 0.5 mg of protein. At higher protein levels the reagent consumption will be seen as decreased Eu/protein yield. Labeling yield can be controlled by choosing appropriate volume or by adjusting labeling times. Avoid using too small volumes where the reaction may be too extensive. Labelling of small molecules Small drug molecules and other molecules can be labeled directly if they contain available amino-group, or may be labeled through a linker arm. This enables the molecule to be used directly in assays without interference from any added chelate. The linker arm can be incorporated during synthesis or be added after synthesis by normal organic chemistry procedures. KA2096 4 / 8
General Information Materials Supplied List of component Component Amount Lyophilized W1024 Chelate (148 nmol) 100 μg Europium standard 0.5 ml Enhancement Solution 50 ml 96 well plate-clear bottom: Unreacted microtitration plate for DELFIA assays. 1 plate 96 well plate-black bottom: Unreacted microtitration plate for FRET assays. 1 plate Note: The plates are used for background and labelling efficiency checks only. The one with the clear bottom is for DELFIA type of assays the black opaque one is for FRET. Storage Instruction The unopened vial of W1024 Chelate is stable for 5 years or more if stored at -20 C. The Enhancement solution should be stored at 4 C and in the dark. Store all other materials at 4 C and in the dark. Materials Required but Not Supplied Labeling Buffer, for most proteins: 50 mm carbonate buffer, ph 9.3 is recommended Elution Buffer, Tris buffer, 50 mm, ph7.5 with 0.5% sodium azide Pipettes For proteins: Gel Filtration columns NAP5, PD-10 or G-25 column can be used for rapid purification. Alternatively a Sephadex G50 column for more complete separation or a combination of gel filtration columns to fractionate proteins (may be needed for separation of aggregates and dimers from single protein is Sepharose 6B plus G50). Spin columns should not be used. Column decontamination buffer, elution buffer with 1 mm EDTA Spectrophotometer (for protein measurement) Shaker Time-resolved fluorometer Precautions for Use The Europium Labeling Kit is intended for research use only, and is not intended for use in human diagnostic or therapeutic procedures. For assay work (whether 96 or 384 or 1536 well formats) Standard clear low fluorescence background plates KA2096 5 / 8
from NUNC. Porvair or Greiner are normally used, but for achieving the best low backgrounds a black plate can be better. KA2096 6 / 8
Protocol Reagent Preparation Preparation of the test Standard Ensure all reagents are at room temperature. Pipette 10 μl of Europium chloride standard into an Eppendorf or similar tube, make up to 1 ml with the Enhancement Solution. This is a stock and can be stored for further use at 4 C. From this stock solution take a 200 μl aliquot and pipette into a well of a 96 well format clear plastic microtitration plate. Using the standard Europium settings read in the instrument. The read out should be approximately 1,000,000 (1 million counts). However it is possible on older instruments for a lower to be recorded. Assay Procedure Pre-treatment of protein to be labeled It is recommended that the buffer be changed to a suitable Labeling Buffer prior to the labeling process to avoid interferences and ph changes. A NAP-5, 10 or equivalent column is recommended. 1. Equilibrate the column with 25 ml of a Labeling Buffer 2. Add the protein solution to the column and rinse with Labeling Buffer (keep the volume as small as possible to be able to adjust labeling volumes). 3. Collect the protein fraction after 2.5 ml (PD-10) void volume. Because the procedure is for X ul volume of pre-treated protein, it is worth collecting the protein in a small a volume as possible. The maximum volume of protein for buffer exchange (1.0 ml for NAP-10) produces 1.5 ml of eluted protein solution. Smaller protein volumes can be eluted with smaller buffer volumes provided that the protein is monitored. Labeling 1 Open the Europium labeling reagent carefully. 2 Add X ul of the protein in the Labeling buffer to the reagent vial. 3 Adjust the ph if needed. 4 Mix gently to dissolve the reagent and incubate overnight at RT. 5. Equilibrate a column with a 3x void volume of elution buffer. If the column has been previously used with Eu labeled proteins it is recommended to wash it with a decontamination buffer prior to equilibration. Add the reaction mixture to the column and rinse the Labeling vial with a small volume of elution buffer and elute with the same buffer. 6. Monitor the eluate by UV-absorbance at 280 nm. Collect fractions of 1 2 ml. 7. Follow Eu elution by measuring fluorescence of the fractions after suitable dilution (e.g. 1/10000 to Tris buffer) KA2096 7 / 8
8. Pool the fractions containing the labeled proteins. When using gel filtration column, avoid pooling aggregated proteins. Note: PD-10 and NAP columns are not able to fractionate proteins based on molecular weight, but can be used for assays where protein aggregation is not likely to cause problems. 9. Measure the total Eu concentration using the Enhancement Solution supplied. Dilute pooled protein fraction in serial dilution into 1/100, 1/10,000 and 1/1,000,000 or into Enhancement Solution. For standardization, dilute 100 nm Eu standard into Enhancement Solution. Shake and let the chelate dissociate for 20 min before measuring the fluorescence with a suitable TR plate reader. Calculate the Eu concentration in the pool. 10. Calculate protein concentration. It can be roughly calculated by estimated yield from added amount. It also can be calculated by UV absorbance taking into account the chelate absorbance at 280 nm (ε = 16,000). For IgG proteins, the concentration can be calculated can be calculated as follows: Protein (mg/ml) = (A280- [Eu] (mm) x 16) / 1.34 11. Calculate the labeling yield Eu/protein 12. Store the protein in elution buffer at + 8 ºC. The storage buffer should not contain chelating agents, acidic ph nor phosphate. If protein concentration is very low, it should be stabilized with a suitable carrier protein. Eu-free bovine serum albumin is most commonly used. Characterization of the labeled protein 1. Measure the total Eu concentration using Enhancement solution supplied. Dilute pooled protein fractions in serial dilution into 1/100, 1/10,000 and 1/11,000,000 into Enhancement Solution. For standardization, dilute 100 nm Eu-standard into Enhancement Solution. Shake & let the chelate dissociate for 20 minutes before measuring with a suitable TR plate reader. Calculate the Eu concentration in the pool. 2. Calculate protein concentration. It can be roughly calculated by estimated yield from added amount. It also can be calculated by UV absorbance taking into account the chelate absorbance at 280 nm (ε = 16,000). For IgG proteins, the concentration can be calculated can be calculated as follows: Protein (mg/ml) = (A280- [Eu] (mm) x 16) / 1.34 Protein (IgG) μm = protein (mg/ml) x 1,000,000/16,000 3. Calculate the Labeling yield Eu/protein 4. Store the protein in Elution buffer at 8 C. The storage buffer should not contain chelating agents, acidic ph nor phosphate. If protein concentration is very low, it should be stabilized with a suitable carrier protein. Eu-free bovine serum albumen is most. Pure gelatin, casein & baby milk powder can also be used. Storage To increase the stability of labeled proteins, a stabilizer (BSA) can be used as a carrier protein at a final concentration of 1.0%. Store labeled proteins at 4 C. KA2096 8 / 8