We thank the Australian Research Council (ARC) for ARC Future Fellowship (FT120100101) and ARC Centre of Excellence CE140100036) grants to P.T. and the Mark Wainwright Analytical Centre at UNSW for access to mass spectrometry and NMR facilities.

Note: The following protocol is designed for the synthesis of a protein-dye bioconjugate as shown in Figure 1. It is a general protocol for the reaction of a maleimide with free surface cysteine containing proteins, with notes inserted where applicable to assist with membrane protein bioconjugates, protein-polymer bioconjugates, and synthetic protein dimer (protein-protein) bioconjugates. In this particular case, the protein iso-1 cytochrome c has one surface cysteine residue available to react which allows a highly specific labelling to occur. If a protein of interest has multiple cysteine residues, the same protocol applies, albeit with the loss of specificity and product homogeneity. Chemistry targeting surface lysine residues, using N-hydroxysuccinimidyl esters or isothiocyanates, may be a simpler approach if specificity is not required.
<img alt="Figure 1" src="/files/ftp_upload/54157/54157fig1.jpg" /> 
Figure 1. Bioconjugation Reaction Scheme. As an example case, a light harvesting, ruthenium-based antenna molecule will be attached to cytochrome c via Michael addition of a pendant maleimide on the ruthenium-based antenna molecule and an exposed cysteine residue (CYS102) on the protein. The red area of the cyt c surface indicates the heme group. <a href="https://www.jove.com/files/ftp_upload/54157/54157fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Purification of Cytochrome c
Note: This step is not applicable to all proteins. However, it is important to know that a protein obtained from a commercial supplier can contain other, undesired protein isoforms which may need to be removed by further purification13.
<ol>
	<li>Dissolve 2.4 g of sodium dihydrogen phosphate (Mw = 120 g mol-1) in 1 L of ultrapure water to prepare a buffer solution containing 20 mM NaH2PO4. Adjust the pH with 1 M NaOH to pH 7. 
	Note: The phosphate buffers used in this protocol should be prepared freshly on a daily basis, and filtered through a 0.2 &#181;m cellulose membrane filter prior to use.</li>
	<li>Dissolve 29.22 g of sodium chloride (Mw = 58.44 g mol-1) in 500 ml of the 20 mM NaH2PO4 buffer to make a 20 mM NaH2PO4 and 1 M NaCl buffer. This is the elution buffer for the purification in step 1.6).</li>
	<li>Dissolve 12.0 mg of lyophilized cytochrome c (cyt c) in 6 ml of pH 7 20 mM phosphate buffer.</li>
	<li>Separately dissolve 14.7 mg of dithiothreitol (DTT, Mw = 154.25 g/mol) in 95.3 &#181;l of ultrapure water to prepare a 1 M stock solution. 
	Note: The DTT stock solution should be prepared freshly, as this reagent is susceptible to oxidative deactivation in aqueous solution.</li>
	<li>Pipette 60 &#181;l of the 1 M DTT solution into the protein solution to reduce the cyt c. The color of the solution will change from dark red to light red upon mixing.</li>
	<li>Filter the reduced protein solution through a 0.22 &#181;m low protein binding PVDF syringe filter before injecting onto any chromatographic media.</li>
	<li>Attach a 3.3 ml strong cation exchange column between the injection loop and UV-Vis detector of a Fast Protein Liquid Chromatography (FPLC) instrument.</li>
	<li>Equilibrate the column with 3 column volumes of ultrapure water, followed by 3 column volumes of 20 mM pH 7 phosphate buffer, using a flow rate of 1 ml/min.</li>
	<li>Load 1 ml of reduced crude cyt c into the injection loop, and start a gradient method from 328 - 450 mM NaCl, over 5 column volumes. Monitor the 280 nm and 410 nm channels of the UV-Vis detector, and collect the largest peak.</li>
	<li>Increase the salt concentration to 1 M for 2 column volumes to elute iso-2 cytochrome c, and after the column has been flushed, re-equilibrate the column with 2 column volumes of 20 mM phosphate buffer before injecting the next crude aliquot.</li>
	<li>Repeat steps 1.9-1.10 until all the crude protein has been purified.</li>
	<li>Pool the iso-1 containing fractions and concentrate them using a 3.5 kDa Molecular Weight Cutoff (MWCO) spin filter and centrifuging at 3,000 x g.</li>
	<li>Load the concentrated protein into 3.5 kDa MWCO dialysis cassettes and dialyze against ultrapure water overnight, with 2 changes of water.</li>
	<li>Determine the concentration of the pure, concentrated protein solution by taking 10 &#181;l, diluting it to 100 &#181;l with ultrapure water, and taking an absorbance spectrum. Use a low volume, 100 &#181;l quartz cuvette to obtain protein absorbance spectra. Typically, the undiluted protein concentration is on the order of 50 - 100 &#181;M.
	<ol>
		<li>Use the characteristic 410 nm peak of cyt c to quantify the concentration using the Beer-Lambert law with a molar absorptivity value of 97.6 cm-1 mM-1: 
		A = &#949;&#160;&#215; c&#160;&#215; &#953; 
		where A is the absorbance,&#160;&#949; is the molar absorptivity, c is the concentration in mM, and&#160;&#953; is the path length of the cuvette in cm13.</li>
	</ol>
	</li>
	<li>At this point, divide the protein into 1 ml aliquots and keep frozen at -20 &#176;C until they are needed. Cyt c can be frozen and thawed without loss of structure or function, but repeat cycles will begin to denature the protein. 
	Note: Proteins tolerate freezing to different degrees. For example, green fluorescent proteins5,9 should not be frozen, instead stored in a refrigerator.</li>
</ol>
2. Synthesis of Cytochrome c Bioconjugates
<ol>
	<li>Dissolve 0.9 mg of ruthenium(II) bisterpyridine maleimide (Ru(II) (tpy)2-maleimide, 0.975 &#956;mol, 6 equivalents) in 600 &#956;l of acetonitrile. 
	Note: If the maleimide used is soluble in water, prepare a stock solution in water instead of acetonitrile. It is important for the maleimide to be soluble in the reaction buffer. Dimethylsulfoxide, N,N-dimethylformamide, and acetonitrile are all commonly used adjuvants to assist in the dissolution of low molecular weight12, often hydrophobic maleimide reactants.</li>
	<li>Prepare a buffer solution containing 100 mM phosphate buffer and 100 mM ethylenediaminetetraacetic acid (EDTA, Mw = 292.24 g/mol), which when diluted to 20 mM is at pH 7. Add solid sodium hydroxide until the EDTA is dissolved (several grams typically) and adjust the pH to 7.</li>
	<li>Dissolve 2.87 mg of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP15, Mw = 286.65 g/mol) in 1 ml of ultrapure water to prepare a 10 mM stock solution. This step is important to ensure that the cysteine on the protein is fully reduced prior to maleimide coupling15. 
	Note: The TCEP stock solution should be prepared freshly for each reaction, as this reagent is susceptible to oxidative deactivation in aqueous solution.</li>
	<li>Combine 11.4 ml of ultrapure water with 3 ml of 100 mM phosphate/EDTA stock solution in a 50 ml plastic tube. When diluted further with protein and maleimide stock solutions the buffer concentration will be 20 mM. 
	Note: If the protein being labelled is a transmembrane protein, ensure that a suitable detergent is added to the buffer to solubilize the protein. If a detergent is not used the membrane protein may slowly precipitate, which will negatively impact reaction yields. Use the detergent in all subsequent purification steps.</li>
	<li>Add 0.15 &#181;mol (3 ml of a 50 &#181;M solution, 1 equivalent) of purified cyt c to the phosphate-EDTA buffer.</li>
	<li>Add 7.5 &#181;l of the TCEP stock solution (0.075 &#181;mol, 0.5 equivalents) to the protein solution and leave stirring for 5 min to reduce any protein that may have dimerized due to cysteine oxidation. 
	Note: Do not add TCEP if the protein of interest does not dimerize readily. Check this by running a protein gel under non-reducing conditions.</li>
	<li>Add 600 &#181;l of the acetonitrile solution of Ru(II) (tpy)2-maleimide to the reduced, buffered solution of cyt c, and leave the reaction mixture stirring, in the dark, at RT, for 24 hr.</li>
	<li>Concentrate the reaction mixture using 3.5 kDa MWCO spin filters, centrifuging at 3,000 x g, repeating 2-3 times with fresh 20 mM phosphate buffer until the filtrate runs clear. Remove as much unreacted maleimide from the reaction mixture as possible before the next stage of purification. 
	Note: At this point, the crude bioconjugation mixture can be stored in the refrigerator, in the dark. It is important to remove unreacted maleimide dye by centrifuge dialysis before storage as the maleimide can slowly and nonspecifically react with lysine residues on the surface of the protein.</li>
</ol>
3. Purification of Cytochrome c Bioconjugates
<ol>
	<li>Dissolve 2.4 g of sodium dihydrogen phosphate and 29.22 g of sodium chloride in 1 L of ultrapure water to prepare a buffer solution containing 20 mM NaH2PO4 and 0.5 M NaCl. This is the running buffer for the immobilized metal-affinity chromatography (IMAC) purification.</li>
	<li>Dissolve 17 g of imidazole (Mw = 68.077 g mol-1) in 500 ml of the 20 mM NaH2PO4 and 0.5 M NaCl buffer. This is the elution buffer for the IMAC purification.</li>
	<li>Adjust the pH of both buffers to pH 7 using 1 M NaOH or HCl, and filter them through 0.22 &#181;m regenerated cellulose membranes before use in the FPLC.</li>
	<li>As the IMAC columns purchased are shipped without any metal ions loaded onto the column, prepare the column for a Ni2+-based purification by washing the column with 3 ml of ultrapure water, 3 ml of 100 mM nickel acetate solution, and 6 ml of ultrapure water. If the column is not to be used immediately wash through 3 ml of 20% ethanol and store the column in the refrigerator to prevent bacterial growth.</li>
	<li>Attach a Ni2+-loaded 1 ml IMAC column to the FPLC between the injection loop and UV-Vis detector.</li>
	<li>Equilibrate the column with 3 column volumes of 20 mM phosphate, 0.5 M NaCl buffer using a flow rate of 0.5 ml/min.</li>
	<li>Load 100 &#181;l of crude reaction mixture (filtered through a 0.22 &#181;m syringe filter) on the Ni2+ IMAC column. Wash the column with 3 column volumes of running buffer to elute non-reacted cyt c, followed by an imidazole gradient from 0 to 125 mM over 3 column volumes to elute the ruthenium bisterpyridine-cytochrome c bioconjugate (Ru(II)-cyt c).</li>
	<li>Wash the column with 250 mM imidazole buffer for 5 column volumes, then re-equilibrate the column with 20 mM phosphate, 0.5 M NaCl and repeat step 3.7 until all the crude bioconjugate has been purified.</li>
	<li>Pool the bioconjugate fractions and concentrate them using a 3.5 kDa MWCO spin filter, centrifuging at 3,000 x g.</li>
	<li>Load the concentrated bioconjugate into 3.5 kDa MWCO dialysis cassettes and dialyze against ultrapure water overnight, with 2 changes of water.</li>
	<li>Determine the concentration of the pure, concentrated bioconjugate solution by UV-Vis, using the same molar absorptivity value as iso-1 cytochrome c (97.6 mM-1 cm-1) at 410 nm. Typically, the bioconjugate concentration is on the order of 50 - 100 &#181;M.</li>
	<li>Divide the bioconjugate into 25 &#181;l aliquots and keep frozen at -20 &#176;C until they are needed.</li>
</ol>
4. Characterization of Cytochrome c Bioconjugates
<ol>
	<li>Determination of bioconjugate mass by MALDI-TOF MS
	<ol>
		<li>Dissolve 10 mg of caffeic acid in 1 ml of an acetonitrile/water/trifluoroacetic acid solution (80:20:0.1, v/v/v).</li>
		<li>Dilute 5 &#181;l of concentrated protein solution with 5 &#181;l of caffeic acid solution.</li>
		<li>Spot 0.5 &#181;l of caffeic acid solution on the MALDI target plate and allow the solution to dry.</li>
		<li>Spot 0.5 &#181;l the sample/matrix solution on top of this caffeic acid spot, allow the spot to dry. Spot another 0.5 &#181;l of sample matrix on top of this to &#34;sandwich&#34; the sample between layers of matrix, and allow to dry.</li>
		<li>Acquire mass spectra in linear mode using suitable instrument settings for proteins16.</li>
	</ol>
	</li>
	<li>Investigation of bioconjugates by gel electrophoresis
	<ol>
		<li>Prepare 10 &#181;l of each protein sample to be run by diluting protein samples with premixed lithium dodecyl sulfate (LDS, pH 8.4) buffer (4x) to a final concentration of approximately 20 &#181;g per well. For wells that require reducing conditions, add 1 &#181;l of 500 mM dithiothreitol (DTT) reducing agent (10x).</li>
		<li>Heat samples at 70 &#176;C for 10 min.</li>
		<li>Add 50 ml of premixed 1 M MES, 1 M Tris base, 2% SDS, 20 mM EDTA (pH 7.7) running buffer mixture (20x) from a commercial source to 950 ml of ultrapure water to prepare the gel running buffer.</li>
		<li>Remove the plastic comb from the gel and place the gel in the gel running tank. Fill up the tank with gel running buffer so that the wells are covered with buffer.</li>
		<li>Load samples carefully onto a precast 12% Bis-Tris, 1 mm, 10-well gel using long pipette tips to assist in loading. Load the prestained 10 protein molecular weight marker (3-188 kDa) into a middle well of the gel to aid analysis.</li>
		<li>Run the gel at 200 V constant voltage for 35 min.</li>
		<li>Stain the gel with a commercial Coomassie blue solution for 2 hr, and wash with ultrapure water for 48 hr.</li>
	</ol>
	</li>
	<li>Determination of bioconjugate purity by UV-Vis spectroscopy
	<ol>
		<li>Prepare 120 &#181;l of 5 &#181;M solutions of Ru(II) (tpy)2-maleimide, iso-1 cyt c, and Ru(II)-cyt c.</li>
		<li>Measure the baseline spectrum of the quartz cuvette containing only ultrapure water, from 250 nm to 650 nm.</li>
		<li>Measure the spectrum of each component, ensuring the cuvette is rinsed and dried between each measurement.</li>
		<li>Plot the absorbance of each component as a function of wavelength, and compare the linear sum of the starting materials with the final product to determine if a 1:1 ratio of Ru(II) (tpy)2-maleimide to cyt c has reacted.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Purification of the starting materials before a bioconjugation is of utmost importance. Proteins obtained from commercial recombinant sources often contain other isoforms of the protein of interest, which can have different surface chemistry and reactivity. For example, in the described bioconjugation, the commercially available cyt c contains a mixture of both iso-1 and iso-2 cyt c12,14,17. Iso-2 and iso-1 forms of cytochrome c are largely homologous, with the main difference being ...

Bioconjugation involves covalently linking one biomolecule with another or with a synthetic molecule such as a dye, drug or a polymer. Protein bioconjugation methods are now extensively used in many chemistry, biology and nanotechnology research groups with applications ranging from fluorescent dye labelling1,2, making of protein (antibody)-prodrugs3 (antibody drug conjugates &#8212; ADCs) synthesis of protein dimers4,5, through to self-assembling protein-polymer hybrids6,7 used in nanomedicine8 and systems chemistry9.
Specificity of the chemistry used for bioconjugation, while not always critical, is of utmost importance for most functional protein bioconjugates, so as to not interfere with the active site of the target protein. The ideal bioconjugation reaction needs to fulfill several criteria, including: i) targeting rare or unique sites on the protein of interest, ii) be selective towards this target, iii) proceed under non-denaturing conditions to avoid protein unfolding and iv) be high-yielding as the target protein is usually only available at sub-millimolar concentration. The maleimide - cysteine Michael addition comes close to fulfilling all these criteria, and has for that reason long claimed a special status in the field of bioconjugate chemistry10. This is because i) many proteins containing only one cysteine residue on their surface can be genetically engineered there, ii) at the correct pH the reaction is highly selective towards cysteine, iii) it proceeds smoothly in aqueous buffers and iv) it is very fast with the second order rate constant of maleimides to cysteine-containing proteins reported to exceed 5,000 M-1 sec-1 in some cases11. Provided the protein of interest can tolerate a small (&#8776; 5-10%) amount of organic co-solvent12, almost any maleimide-functionalized dye, polymer, surface or another protein can be linked to proteins. In addition, maleimides are more specific for cysteines on proteins than iodoacetamides, which are more prone to reacting with other nucleophiles at elevated pH; and more stable than disulfide-based conjugations which need to be kept at acidic pH to prevent disulfide exchange13.
Here we report a generic protocol for the conjugation of maleimide-functionalized molecules to a protein containing a single cysteine residue using the reaction between a Ru(II)-based chromophore and the redox protein cytochrome c as an example. This protocol is equally applicable to most other proteins containing an accessible surface cysteine residue and the corresponding maleimide-functionalized target, be it another protein, a fluorescent dye, a chromophore or a synthetic polymer.

<table border="0" cellpadding="0" cellspacing="0" width="913">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium dihydrogen phosphate</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">71496</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium hydroxide</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">71691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium chloride</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">73575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">cytochrome c, from saccaromyces cerevisiae</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">C2436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">dithiothreitol</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">TSKgel SP-5PW</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">Tosoh SP-5PW, 07161</td>
			<td>3.3 mL strong cation exchange column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15&#160;</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;">UFC900308</td>
			<td>3.5 kDa spin filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide-A-Lyzer mini dialysis units</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">66333</td>
			<td>3.5 kDa dialysis cassetes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ru(II) bisterpyridine maleimide</td>
			<td style="width:196px;">Lab made</td>
			<td style="width:151px;"></td>
			<td>see ref (14)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">acetonitrile</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">A3396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">ethylenediaminetetraacetic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">03609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tris(2-carboxyethyl)phosphine hydrochloride&#160;</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">93284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">imidazole</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">56749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">nickel acetate</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">244066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">AcroSep IMAC Hypercell column</td>
			<td style="width:196px;">Pall</td>
			<td style="width:151px;">via VWR: 569-1008</td>
			<td>1 mL IMAC column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.2 micron cellulose membrane filter</td>
			<td style="width:196px;">Whatman</td>
			<td style="width:151px;">Z697958</td>
			<td>47 mm filter for buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.2 micron PVDF membrane filter</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;">SLGV013SL</td>
			<td>syringe filters for proteins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">hydrochloric acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">84426</td>
			<td>extremely corrosive! Use caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">caffeic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">60018</td>
			<td>MALDI matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">trifluoroacetic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">91707</td>
			<td>extremely corrosive! Use caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SimplyBlue SafeStain</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">LC6060</td>
			<td>Coomassie blue solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE Novex 12% Bis-Tris Gel</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0342BOX</td>
			<td>precast protein gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">SeeBlue Plus2 Pre-stained Protein Standard</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">LC5925</td>
			<td>premade protein ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE LDS Sample Buffer (4X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0008</td>
			<td>premade gel sample buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE Sample Reducing Agent (10X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0004</td>
			<td>premade gel reducing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE MES SDS Running Buffer (20X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0002</td>
			<td>premade gel running buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Voyager DE STR MALDI reflectron TOF MS</td>
			<td style="width:196px;">Applied Biosystems</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Acta FPLC</td>
			<td style="width:196px;">GE</td>
			<td style="width:151px;"></td>
			<td>Fast Protein Liquid Chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Cary 50 Bio Spectrophotometer</td>
			<td style="width:196px;">Varian-Agilent</td>
			<td style="width:151px;"></td>
			<td>UV-Vis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Milli-Q ultrapure water dispenser</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;"></td>
			<td>ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Low volume UV-Vis Cuvette</td>
			<td style="width:196px;">Hellma</td>
			<td style="width:151px;">105-201-15-40</td>
			<td>100 microliter cuvette</td>
		</tr>
	</tbody>
</table>

synthesis of protein bioconjugates via cysteine-maleimide chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

The synthesis of bioconjugates is confirmed by three primary methods: Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), polyacrylamide gel electrophoresis, and Ultraviolet-Visible (UV-Vis) spectroscopy, as shown in Figures 2, 3 and 4. A mass increase corresponding to the mass of the appended small molecule, and the lack of an unreacted protein demonstrates the successful covalent linkage of Ru(II) (tpy)2</su...

Watch this Scientific Journal Video about Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry at JoVE.com

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

synthesis-of-protein-bioconjugates-via-cysteine-maleimide-chemistry

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38.2K 조회수.  The University of New South Wales. This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

비디오: Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides (CHIPS)

The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order to streamline synthesis by saving steps, time and material compared to conventional methods that require the introduction and removal of activating or directing groups. Therefore, the functionalization of C-H bonds is also attractive for green chemistry. Under oxidative conditions, two C-H bonds or one C-H and one heteroatom-H bond can be transformed to C-C and C-heteroatom bonds, respectively. Often these oxidative coupling reactions require synthetic oxidants, expensive catalysts or high temperatures. Here, we describe a two-step procedure to functionalize indole derivatives, more specifically tetrahydrocarbazoles, by C-H amination using only elemental oxygen as oxidant. The reaction uses the principle of C-H functionalization via Intermediate PeroxideS (CHIPS). In the first step, a hydroperoxide is generated oxidatively using visible light, a photosensitizer and elemental oxygen. In the second step, the N-nucleophile, an aniline, is introduced by Br&#248;nsted-acid catalyzed activation of the hydroperoxide leaving group. The products of the first and second step often precipitate and can be conveniently filtered off. The synthesis of a biologically active compound is shown.

Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides CHIPS

A two-step procedure for the synthesis of pharmaceutically active indole-derivatives by C-H functionalization with anilines is described, using photo- and Br&#248;nsted acid catalysis.

중간 과산화물을 통해 광화학 및 산 - 촉매 CH 기능화에 의한 항 바이러스 Tetrahydrocarbazole 유도체의 합성 (CHIPS)

The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order ...

연구

화학

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

기능화의 합성 및 특성 금속 - 유기 프레임 워크

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Synthesis and Reaction Chemistry of Nanosize Monosodium Titanate

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the material with Au(III) ions. The synthesis method was derived from a sol-gel process used to produce micron-sized monosodium titanate (MST), with several key modifications, including altering reagent concentrations, omitting a particle seed step, and introducing a non-ionic surfactant to facilitate control of particle formation and growth. The resultant nMST material exhibits spherical-shaped particle morphology with a monodisperse distribution of particle diameters in the range from 100 to 150&#160;nm. The nMST material was found to have a Brunauer-Emmett-Teller (BET) surface area of 285 m2g-1, which is more than an order of magnitude higher than the micron-sized MST. The isoelectric point of the nMST measured 3.34 pH units, which is a pH unit lower than that measured for the micron-size MST. The nMST material was found to serve as an effective ion exchanger under weakly acidic conditions for the preparation of an Au(III)-exchange nanotitanate. In addition, the formation of the corresponding peroxotitanate was demonstrated by reaction of the nMST with hydrogen peroxide.

The surfactant mediated sol-gel synthesis of nanosized monosodium titanate is described, along with preparation of the corresponding peroxide modified material. An ion-exchange reaction with Au(III) is also presented.

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the ...

The Synthesis of [Sn10(Si(SiMe3)3)4]2- Using a Metastable Sn(I) Halide Solution Synthesized via a Co-condensation Technique

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a sterically demanding ligand, has increased in recent years. The metastable Sn(I) halide is synthesized at&#160;&#34;outer space conditions&#34; via the preparative co-condensation technique. Thereby, the subhalide is synthesized in an oven at high temperatures, around 1,300 &#176;C, and at reduced pressure by the reaction of elemental tin with hydrogen halide gas (e.g., HCl). The subhalide (e.g., SnCl) is trapped within a matrix of an inert solvent, like toluene at -196 &#176;C. Heating the solid matrix to -78 &#176;C gives a metastable solution of the subhalide. The metastable subhalide solution is highly reactive but can be stored at -78 &#176;C for several weeks. On heating the solution to room temperature, a disproportionation reaction occurs, leading to elemental tin and the corresponding dihalide. By applying bulky ligands like Si(SiMe3)3, the intermediate metalloid cluster compounds can be trapped before complete disproportionation to elemental tin. Hence, the reaction of a metastable Sn(I)Cl solution with Li-Si(SiMe3)3 gives [Sn10(Si(SiMe3)3)4]2- 1 as black crystals in high yield. 1 is formed via a complex reaction sequence including salt metathesis, disproportionation, and degradation of larger clusters. Further, 1 can be analyzed by various methods like NMR or single crystal X-ray structure analysis.

The Synthesis of [Sn10SiSiMe334]2- Using a Metastable SnI Halide Solution Synthesized via a Co-condensation Technique

The disproportionation reaction of a metastable Sn(I) chloride solution, obtained via the preparative co-condensation technique, is used for the synthesis of a metalloid tin cluster compound.

합성 [주석<sub&gt; (10)</sub&gt; (시 (SIME<sub&gt; 3</sub&gt;)<sub&gt; 3</sub&gt;)<sub&gt; 4</sub&gt;]<sup&gt; 2</sup<sup&gt; -</sup&gt; 준 안정 주석 (I)을 공 축합 기법을 통해 할라이드의 합성 용액을 사용하여

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

2&#39;- 포스 핀 올리고 뉴클레오타이드를 함유하는 올리고머의 고상 합성을위한 프로토콜<em&gt; O</em&gt; 원형 이색 성을 통한 - 티오 페닐 메틸 변형 및 특성 분석

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

활성 표면 성장 메커니즘을 통해 기판 바인딩된 Au 나노 와이어의 합성

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

Au, Pd, Pt Aerogels 직접 솔루션 기반 감소를 통해 빠른 합성 방법

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

이기에 친 환 경적 Steglich 에스테 르 화를 통해 에스테 르의 합성

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

대장균-셀 무료 단백질 합성 기반: 강력 하 고 유연 하며 액세스할 수 있는 플랫폼 기술에 대 한 프로토콜

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock copolymers are produced by one of three techniques: thin film rehydration, solvent switching or polymerization-induced self-assembly, and produce only flexible, polydisperse cylinders. Crystallization-driven self-assembly (CDSA) is a method which can produce cylinders with these properties, by stabilizing structures of a lower curvature due to the formation of a crystalline core. However, the living polymerization techniques by which most core-forming blocks are formed are not trivial processes and the CDSA process may yield unsatisfactory results if carried out incorrectly. Here, the synthesis of cylindrical nanoparticles from simple reagents is shown. The drying and purification of reagents prior to a ring-opening polymerization of &#949;-caprolactone catalyzed by diphenyl phosphate is described. This polymer is then chain extended by methyl methacrylate (MMA) followed by N,N-dimethyl acrylamide (DMA) using reversible addition&#8722;fragmentation chain-transfer (RAFT) polymerization, affording a triblock copolymer that can undergo CDSA in ethanol. The living CDSA process is outlined, the results of which yield cylindrical nanoparticles up to 500 nm in length and a length dispersity as low as 1.05. It is anticipated that these protocols will allow others to produce cylindrical nanostructures and elevate the field of CDSA in the future.

Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of &#949;-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined.

생분해성 블록 공합체의 결정화 구동 자가 조립을 통해 단분산 원통형 나노 입자의 합성

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock ...

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