To obtain human Oct-1Z-specific antibodies, rabbits were immunized with the Oct-1Z isoform N-end-specific synthetic peptide MKTRMKIFVMIHFHLMNS conjugated to PEGilated KLH using the Imject™ Maleimide-Activated mcKLH Spin Kit (Thermo Scientific™) according to the manufacturer's protocol. The Thermo Scientific Imject Maleimide-Activated mcKLH Spin Kit contains stabilized lyophilized Maleimide-Activated mcKLH (in PBS with stabilizer) and accessory components enabling easy production of the ready-to-use immunogens for subsequent immunization. Rabbits were immunized s.c. with the five doses containing 250 µg of conjugate (peptide-KLH) each. Blends were obtained nine days after the third and the last dose. The obtained antibodies were precipitated with ammonium sulfate and purified from the anti- KLH antibodies using affinity columns. To avoid cross-reactivity, anti-Oct-1Z antibodies were let through the affinity column with the bound Oct-1L and Oct-1A-specific peptides.
Imject maleimide activated mcklh spin kit
Manufactured by Thermo Fisher Scientific
The Imject Maleimide-Activated mcKLH Spin Kit is a laboratory product designed for the maleimide activation of carrier proteins, such as mcKLH (Megathura crenulata keyhole limpet hemocyanin). The kit provides a convenient and efficient method for the covalent conjugation of molecules, such as peptides or haptens, to the activated carrier protein.
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Lab products found in correlation
Melon gel igg purification kit, by Thermo Fisher Scientific (1 mentions)
Titermax gold, by Merck Group (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Ultracel 10 membrane, by Merck Group (1 mentions)
Mouse anti α tubulin dm1a, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti gst, by Cell Signaling Technology (1 mentions)
Mouse anti myc 9b11, by Cell Signaling Technology (1 mentions)
Sulfolink immobilization kit for peptides, by Thermo Fisher Scientific (1 mentions)
4 protocols using imject maleimide activated mcklh spin kit
1
Generation of Oct-1Z-Specific Antibodies
2
Nitrated Peptide Synthesis and Characterization
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Peptides were designed to encompass nitrated portions of calmodulin (nY99 and nY138) and 14-3-3 (nY133). The peptide for 14-3-3- nY133 BMHI shared a sequence identity of 61.9% with 14-3-3 nitroTyr human isoform b. A peptide for general nitration was also designed, containing multiple nitroTyr flanked by β-sheets. Peptides were designed by following the described parameters for Nb generation against a folded protein (Trier et al., 2012 (link)) and were submitted to B-epitope predictors (http://crdd.osdd.net/raghava/abcpred/ ) to determine epitope effectiveness. The peptides contained a N-terminal cysteine to facilitate keyhole limpet hemocyanin (KLH) conjugation. The peptides were synthesized by Genscript, resuspended in TBS and conjugated to KLH with the Thermo Scientific Imject Maleimide-Activated mcKLH Spin Kit by following the manufacturer’s instructions.
3
Synthesis and Purification of Phosphorylated KIND2 Antibody
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The phosphorylated peptide containing the CDK1 consensus motif of KIND2 (GSGSIYSS(H3O4P) PGLYSKT) was synthesized in-house. The peptide was conjugated with keyhole limpet haemocyanin using an Imject Maleimide-Activated mcKLH Spin kit (77666, Thermo Fisher) according to the manufacturer’s protocol. The conjugated immunogen was then mixed with adjuvant (TiterMax Gold, Sigma) and injected into rabbits. The procedure was repeated twice every 3 weeks. Blood was collected 2 weeks after the last injection. IgG was purified using a Melon Gel IgG Purification kit (45212, Thermo Fisher) according to the manufacturer’s protocol. For WB or immunostaining, the non-phosphorylated KIND2 peptide (GSGSIYSSPGLYSKT) was added to the antibody solution in a final concentration of 5 µg ml–1 to prevent binding to non-phosphorylated KIND2.
4
Antibody Generation and Characterization for Phosphorylated FE65
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Rabbit anti-FE65 and rabbit anti-APP A5137 were as described5 (link). Goat anti-FE65 E20 and mouse anti-α-tubulin DM1A were obtained from Santa Cruz. Mouse anti-myc 9B11, mouse anti-GFP JL8, mouse anti-HA 12CA5, rabbit anti-GST and mouse anti-APP 22C11 were purchased from Cell Signaling Technology, Clontech, Roche, Sigma and Millipore, respectively.
The phospho-specific antibody against phosphorylated FE65 at T579 (pT579 FE65) was generated by immunizing rats with a synthetic FE65 phosphopeptide (amino acids 575–586; REQW(pT)PSHVSVC) (GenScript) which contains a phosphorylated T579. A cysteine (C) residue was introduced to the peptide C-terminus for carrier protein conjugation and purification column coupling. Phosphopeptide conjugation was performed using the Imject Maleimide Activated mcKLH Spin kit (ThermoFisher Scientific). The immunized rat sera were purified by the non-phosphopeptide (REQWTPSHVSVC) and then phosphopeptide coupled columns prepared by using a SulfoLink Immobilization kit for peptides (ThermoFisher Scientific). The antibody was eluted and dialyzed against PBS/0.05% sodium azide, and then concentrated by using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane (Millipore).
The phospho-specific antibody against phosphorylated FE65 at T579 (pT579 FE65) was generated by immunizing rats with a synthetic FE65 phosphopeptide (amino acids 575–586; REQW(pT)PSHVSVC) (GenScript) which contains a phosphorylated T579. A cysteine (C) residue was introduced to the peptide C-terminus for carrier protein conjugation and purification column coupling. Phosphopeptide conjugation was performed using the Imject Maleimide Activated mcKLH Spin kit (ThermoFisher Scientific). The immunized rat sera were purified by the non-phosphopeptide (REQWTPSHVSVC) and then phosphopeptide coupled columns prepared by using a SulfoLink Immobilization kit for peptides (ThermoFisher Scientific). The antibody was eluted and dialyzed against PBS/0.05% sodium azide, and then concentrated by using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane (Millipore).
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