An overlapping peptide library spanning the entire length HCMV gO polypeptide from strain TR was obtained from Genscript Biotech, Piscataway, NJ. The peptides consisted of 20 amino acids in length with 7 amino acid overlap and a biotin moiety conjugated to the C-terminus. A panel of 8 a.a. peptides overlapping by 7 a.a. and a biotin moiety conjugated to the C-terminus that were derived from the protein sequences: 248-NTMRKLKRKQAPVKEQSEKK-267 or 326-LRDLATWVYTTLRYRQNPFC-345 (S2 Table ) were also obtained from Genscript Biotech. Peptides derived from the gO polypeptide sequence 235-FRVPKYINGTKLKNTMRKLKRKQAPVKEQSEKK-267 (peptide 19/20 from overlapping library) and 326-LRDLATWVYTTLRYRQNPFC-345 (peptide 26 from overlapping library) were synthesized conjugated onto ovalbumin (ova). These ova-peptides were used to produce New Zealand White rabbit polyclonal sera by priming rabbits with ova-peptides in Freund’s adjuvant then boosting the rabbits with ova-peptides in TiterMax Gold adjuvant (Sigma, St. Louis, MO). Rabbit antibodies specific for peptide 20, 26 and ovalbumin were purified using peptides ova-20 and ova-26 or ova (without peptides) by coupling these proteins to AminoLink (Thermo Scientific).
Aminolink
Manufactured by Thermo Fisher Scientific
Sourced in Germany
AminoLink is a resin-based affinity chromatography product designed for the immobilization of proteins, peptides, and other ligands containing primary amine groups. It provides a simple and efficient way to covalently couple amine-containing molecules to a solid support for subsequent purification or analysis applications.
Automatically generated - may contain errors
8 protocols using aminolink
1
Mapping HCMV gO Antibody Epitopes
2
Immunogenic Epitope Identification and Antibody Generation
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The amino acid sequence of rMBPPfCox11Ct was used to identify immunogenic epitopes with Predict7 software [40 (link)]. The peptide sequence KIQF(Abu)F(Abu)EEQMLNAKEEM where internal cysteines were replaced with alpha aminobutyric acid (Abu), was synthesized by GeneScript, USA. The C-terminal cysteine residue was added for m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) coupling to rabbit serum albumin [41 (link)]. Two Hy-line brown laying hens were immunized with the peptide-carrier conjugate equivalent to 200 µg peptide or 50 µg affinity purified rMBPPfCox11Ct per immunization, emulsified with Freund’s complete adjuvant for the first immunization and Freund’s incomplete adjuvant for three subsequent immunizations at 2-week intervals. Chicken IgY from the yolks of eggs collected 4–16 weeks after the first immunization, was isolated using the PEG6000 precipitation method [42 (link)]. The anti-peptide IgY and anti-rMBPPfCox11Ct IgY was affinity purified using a peptide or rMBPPfCox11Ct affinity column prepared by coupling the respective molecules to a Sulfolink™ or Aminolink™ resin according to the manufacturer’s instructions (Thermo Fisher Scientific).
3
CPDB Treatment of Purified PrP
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CPDB treatment was performed according to Li et al (66 ). Briefly, to purify PrP from various cell lines, fresh cell lysate was prepared and affinity-purified with 100 μg of mAb 8B4 bound to 100 μl of profound co-immunoprecipitated agarose beads (Aminolink, Thermo Fisher Scientific) according to the manufacturer’s instructions. Then, 400 μl of cell lysate was added to the column containing the agarose resin coupling with Abs overnight at 4 °C. The resin was washed six times with ice-cold lysis buffer at 4 °C by centrifuging at 1000g for 1 min. After that, 100 μl of elution buffer was added. After 5 min incubation at room temperature (RT), PrP was eluted by centrifuging at 1000g for 1 min and quickly neutralized in Tris-glycine (pH9.4), which was then subjected to CPDB treatment (0.5 unit/20 μl of eluted PrP) at RT for different periods of time. The samples were then separated on SDS-PAGE and immunoblotted with 4H2.
4
Affinity Purification of Anti-Superantigen Antibodies
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SEA, SEB and TSST-1 were coupled to agarose beads (1 mg SAg/mL of bead volume) of an Aminolink® plus immobilization column (ThermoScientific, Rockford, IL) following the manufacturer’s protocol. Affinity purification of specific antibodies from commercial IVIG (Omrix Biopharmaceuticals, Nes-Ziona, Israel) was carried out according to manufacturer’s protocol with minor modifications: 50 mL of IVIG was incubated with toxin-coupled beads for 90 minutes at room temperature (RT) with gentle rocking, centrifuged, and the supernatant removed and a fresh 50 mL of IVIG incubated with the beads for another 1 hour and 30 minutes. Elution was performed with glycine HCl pH 2.5 buffer. Eluted fractions were collected in neutralizing buffer, containing 0.1 M Tris at pH 9 (final pH 6–7). 37.5 µl of affinity-purified anti-SEA, -SEB, - TSST-1, in semi-log dilutions (0.02–20 µg/ml) or IVIG in semi log dilutions (2.5–2500 µg/ml) and 37.5 µl of a 1 ng/ml preparation of either SEB, SEC. 1–3, SEE, SEH, SEI, SEK, TSST-1, SpeC, or 2 ng/ml of SED, or 3 ng/ml of SpeA were mixed. To test the synergistic activity of purified polyclonal Abs a combination of anti-SEA, -SEB, and –TSST-1 were used in a semi log dilution ranging from 0.02 to 20 µg/ml along with the same amount of toxin as above.
5
Isolation and Purification of sCD177
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Plasma was isolated from full blood of CD177-positive donors. sCD177 was purified from plasma by using affinity chromatography using 1.5 mg mAb 7D8 coupled on a column (AminoLink, Thermo Fisher Scientific, Bonn, Germany), and purified protein was dialyzed in 20 mmol/L Tris-buffered saline buffer, pH 7.4. Protein concentration was determined by using bicinchoninic assay (Thermo Fisher Scientific).
6
Covalent Immobilization of Abl2 SH2
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AminoLink (Thermo Fisher Scientific) beads were used to covalently link Abl2 SH2 domain following purification (9 (link)). Briefly, proteins were gently rotated with AminoLink beads overnight. About 50 mM sodium cyanoborohydride was added to catalyze the reaction. Protein was linked at a final reaction concentration of 1 μM, and the remaining reactive sites on protein-linked beads were blocked with 1 M Tris–HCl, pH 8.0, and 100 mg/ml bovine serum albumin, washed, and stored in assay buffer.
7
Peptide-specific Antibody Enrichment
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Ova-conjugated peptides 20, 26 or ovalbumin were immobilized to agarose (Amino-Link, ThermoFisher) and then incubated with pooled human sera that has been previously described [48 (link)]. The bound antibodies were eluted in 0.1M glycine-pH 2.2 and quickly neutralized with 1/10th volume of 0.1M Tris-pH 9.0 and then buffer exchanged into Tris-Saline pH-7.2 using Zeba buffer exchange columns (ThermoFisher). Antibodies were quantified by SDS-PAGE followed by coomassie staining.
8
Rabbit Immunization with XLR3 Peptide
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The first 18 residues common to both XLR3A and XLR3B, and one amino acid difference from XLR3C, were used for peptide synthesis in rabbit immunization by Biosynthesis Inc. Following initial immunization, two rabbits received five biweekly boosters. Affinity purification of total IgG sera was performed using an AminoLink resin column (Thermo) and purified antibodies were complemented with pre-immune sera.
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