TITLE: Derivitization of polyphosphate with cystamine to facilitate secondary labeling with biotin
AUTHORS: Catherine J. Baker, Stephanie A. Smith, James H. Morrissey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Investigation of the biological roles of inorganic polyphosphate has been facilitated by our previous development of a carbodiimide-based method for covalently coupling primary amine-containing molecules to the terminal phosphates of polyphosphate (Choi et al., Biochemistry 49:9935, 2010). We now extend that approach by using readily available “bridging molecules” containing a primary amine and an additional reactive moiety, including another primary amine, a thiol or a click chemistry reagent such as dibenzocyclooctyne. This two-step labeling method is used to covalently attach commercially available derivatives of biotin, peptide epitope tags, and fluorescent dyes to the ends of polyphosphate. This protocol specifically describes the labeling of heterogenous long-chain polyP with cystamine and the subsequent addition of maleimide-biotin.</div></div>

[STEPS]
?. Prepare and combine all materials in this order to a 1.5, 2 or 5 mL tube.  ABC1     Volume (µL)Final Concentration2Water6593500 mM MOPS pH 8.0200100mM41M polyP (mode chain length: 1000)50100 µM ends (50 mM phosphate monomer)5500 mM Cystamine168 mM (80x ends)62M EDAC75150mM7total1000 At the last minute, dissolve dry EDAC powder in water to make a 2 M solution. Then add to reaction.NOTE:  EDAC is very hydroscopic and will degrade with exposure to humidity. Store powder aliquots of EDAC at -20 °C, dessicated. For each reaction, remove aliquot from freezer, allow to warm to room temperature. Refrain from dissolving until immediately before use. Do not store dissolved EDAC.
ABC1     Volume (µL)Final Concentration2Water6593500 mM MOPS pH 8.0200100mM41M polyP (mode chain length: 1000)50100 µM ends (50 mM phosphate monomer)5500 mM Cystamine168 mM (80x ends)62M EDAC75150mM7total1000
?. Add TCEP to a final concentration of 5 mM (dissolve 5.5 mg TCEP in 191 μL water to make 100mM solution, then add 50 μL to 1000 μL of polyP reaction solution).
?. Immediately incubate at 37 °C for 1 h, agitating occasionally.
?. Stop reaction on ice.
?. Centrifuge briefly to remove any condensation from the top of the tube.
?. Follow manufacturer's instructions for 5 mL Zeba™ Spin Desalting Columns  (7K MWCO), exchanging the buffer into 20 mM Hepes-NaOH pH 7.4 + 5 mM EDTA to remove excess cystamine and to prepare for the upcoming maleimide reaction.
?. Remove an aliquot to quantify the phosphate using malachite green quantification assay using standards diluted in the same buffer used in the previous step.See the paper
?. Using the results of the malachite green and biotin quantitation,  we can get a rough estimate of the degree of labeling(concentration of monophosphate from malachite green)/(mode polymer length)= concentration of polymer(concentration of polymer) *2 = concentration of polyphosphate ends(concentration of biotin)/(concentration of ends) = ratio of biotin to endsNOTE: because polyP is heterogeneous, this calculation is based on the mode chain length, and is merely an approximation.It's not uncommon that your ratio of biotin to ends might be >1. Polymers chains shorter than the mode can cause this. However, if the ratio is way higher (>10, for example) it may be worth confirming the purification step to remove excess reactant biotin is working properly  (Step 11) .
?. Rotate the tube at room temperature for 1.5 h.
?. Follow manufacturer's instructions for 5 mL Zeba™ Spin Desalting Columns  (7K MWCO), exchanging the buffer into 20 mM Hepes-NaOH pH 7.4 + 5 mM EDTA to remove TCEP.
?. Dissolve 6.5 mg maleimide-biotin in 50 uL water to make a 250 mM solution.Combine 32 μL of the 250 mM maleimide-biotin solution to 1000 μL of purified, reduced polyP-cystamine sample to make a 8 mM maleimide biotin solution.
?. If desired, quantify the concentration of biotin using the Pierce Fluorescence Biotin Quantitation Kit according to the manufacturer's instructions.
?. Vortex briefly then rotate at room temperature for 1.5 h.
?. Follow manufacturer's instructions for 5 mL Zeba™ Spin Desalting Columns  (7K MWCO), exchanging the buffer into 20 mM Hepes-NaOH pH 7.4 + 5 mM EDTA to remove excess biotin.Note: this buffer can be replaced depending on the requirements of future experiments. However, for EDAC-labeled polyP we suggest storage in a buffer with a  pH between 7 and 8 and at least 100 µM EDTA to chelate trace metals.