Purified ptfV cysteine variants were reduced with Pierce Immobilized TCEP disulfide reducing gel (Thermo Fisher Scientific) at room temperature for 45 minutes, as per the manufacturer’s instructions. Reduced samples were separated from the TCEP gel by centrifugation using a spin cup paper filter and then labeled with 20-fold molar excess of maleimide-PEG40K (Merck) or EZ-Link maleimide-PEG11-biotin (922.09 Da; Thermo Fisher Scientific) at 4°C for 2 hours. Maleimide-PEG40K–labeled samples were used to estimate labeling efficiency by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE; Table 1 ; supplemental Figure 1). Maleimide-PEG11-biotin–labeled samples were applied to a Q Sepharose column (Cytiva; 1 mL) followed by dialysis to remove excess labeling reagent. Peak fractions containing both the mixture of labeled and unlabeled ptfV were pooled and dialyzed into a buffer of 100 mM of potassium phosphate and 150 mM of NaCl (pH 7.2) overnight at 4°C. Dialyzed samples were applied to a column packed with streptavidin mutein resin (Roche) to separate biotinylated from unbiotinylated protein, as per the manufacturer’s instructions (supplemental Figure 2). Peak fractions containing biotinylated ptfV were concentrated and dialyzed overnight into a buffer of 20 mM of Tris (pH 7.4), 150 mM of NaCl, and 2 mM of calcium chloride at 4°C.
Ez link maleimide peg11 biotin
Manufactured by Thermo Fisher Scientific
Sourced in United States
EZ-Link Maleimide-PEG11-Biotin is a heterobifunctional crosslinker that contains a maleimide group and a biotin group separated by an 11-unit polyethylene glycol (PEG) spacer. The maleimide group can be used to modify thiol-containing molecules, while the biotin group can be used to detect or purify the modified molecules.
Automatically generated - may contain errors
Lab products found in correlation
Q sepharose column, by Cytiva (1 mentions)
Pierce immobilized tcep disulfide reducing gel, by Thermo Fisher Scientific (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Alexafluor 633 c5 maleimide, by Thermo Fisher Scientific (1 mentions)
Texas red c2 maleimide, by Thermo Fisher Scientific (1 mentions)
Fluorescein 5 maleimide, by Thermo Fisher Scientific (1 mentions)
K2hpo4, by Carl Roth (1 mentions)
Nah2po4, by Merck Group (1 mentions)
Amicon ultra 0.5 ml centrifugal filter, by Merck Group (1 mentions)
6 protocols using ez link maleimide peg11 biotin
1
Purified ptfV Cysteine Variant Labeling
2
Biotin-Labeled TMV Nanocarrier Synthesis
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A TMV variant (TMVCys) containing a S3C mutation close to the N-terminus of every CP was employed as a viral enzyme nanocarrier with a spacing of 2.5 to 3.5 nm of coupling sites on their outer protein coat [67 (link)]. This mutant exposed 2130 cysteine residues on each TMV particle presenting surficial sulfhydryl groups. TMVCys extracted from infected Nicotiana tabacum leaves were equipped with PEG11-biotin moieties via maleimide-sulfhydryl-coupling (EZ-Link® Maleimide-PEG11-Biotin, Thermo Scientific, Rockford, IL, USA) according to [56 (link)]. Briefly, a maleimide-PEG11-biotin linker was incubated with TMVCys particles in a molar ratio of 3:1 (linker/CP) for 3 h at 26 °C under agitation. Unbound linker molecules were removed by centrifugal ultrafiltration (Amicon Ultra, 30 kDa molecular weight cut-off, Merck-Millipore, Darmstadt, Germany) in five consecutive washing steps with 10 mM of sodium-potassium-phosphate (SPP) buffer (pH 7.0). The resulting biotinylated TMVCys (TMVCys/Bio) particles were resuspended and stored in this buffer at 4 °C. The particles were analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and colloidal Coomassie Brilliant Blue G-250 staining [68 (link)]. Densitometric comparison of the signals corresponding to the biotinylated and the non-modified form of the CP confirmed biotinylation of >90 % of the CPs.
3
Protein Labeling with Maleimide Tags
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The maleimide-containing
molecules used as tags were fluorescein-5-maleimide (Thermo), Texas
Red C2 maleimide (Thermo), Alexa Fluor 633 C5 maleimide (Thermo),
EZ-link maleimide-PEG11-biotin (Thermo), Histidine6 maleimide (Biomatik),
5′-maleimide 3PolyA (Biosynthesis).
molecules used as tags were fluorescein-5-maleimide (Thermo), Texas
Red C2 maleimide (Thermo), Alexa Fluor 633 C5 maleimide (Thermo),
EZ-link maleimide-PEG11-biotin (Thermo), Histidine6 maleimide (Biomatik),
5′-maleimide 3PolyA (Biosynthesis).
4
Modifying TMV Virus Particles for Biosensing
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As a model plant virus particle, we use a genetically modified TMV variant with a cysteine residue exposed on each coat protein [48 ], which was already utilized in our previous works for biosensing purposes as nano-scaffold for high-density immobilization of enzymes on a sensor surface [18 –20 , 41 ]. Bifunctional maleimide-polyethylenglycol-biotin linkers (EZ-Link Maleimide-PEG11-Biotin (Thermo Scientific, Rockford, IL, USA)) are covalently bound to the thiol groups of these cysteine sites (for details of TMV isolation and biotinylation, see [19 , 20 , 49 ]). The TMV stock solution with a concentration of 125 nM (5 mg/mL) in 10 mM sodium-potassium-phosphate buffer (SPP, 10 mM NaH2PO4 (Merck, Darmstadt, Germany) and 10 mM K2HPO4 (Carl Roth, Karlsruhe, Germany), pH 7) was stored at 4 °C. For ZP measurements, the TMV stock solution was diluted with 10 mM SPP (15 mM ionic strength) to a concentration of 0.1 mg/mL (2.5 nM) and adjusted to different pH values between pH 7 and pH 3 by titration with 1 M HCl (Sigma-Aldrich, Darmstadt, Germany). For the TMV loading experiments, differently concentrated TMV solutions were prepared by diluting the TMV stock solution with 0.1 M SPP buffer, pH 7, and an ionic strength of 0.15 M.
5
Labeled Drosophila Protein Analysis
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Adapted from Vitu et al. (2010) (link), 10 adult Drosophila heads or one plate of transfected cells (expressing pAc5.1B -hhNHA with or without cysteine substitutions) were homogenized with Laemmli Buffer (-βME, +Protease Inhibitor), placed at 95°C for 5 min, spun down (1000× g , 3 min), and had supernatant transferred to new tubes. TCEP (Sigma Aldrich) was added to each sample (final, 5 mM) for 10 min at 23°C. Sample pH was equilibrated to ∼7 using filtered 1 M KOH and EZ-Link Maleimide-PEG11-Biotin (Thermo Fisher) (or DMSO alone) was added (final, 1 mM). Reactions proceeded for 2 min before being quenched with βME (final, 5%) and allowed to sit for 5 min at 23°C. Samples were stored at –80°C.
6
Biotinylation and Purification of Cysteine-containing Helix Protein
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