Five BALB/C mice raised in the Animal Culture Centre of Xiamen University were immunized with the collected recombinant SpHyastatin to generate the antiserum. The initial immunization was subcutaneously injected with 200 μg of SpHyastatin mixed with Freund’s complete adjuvant. The second injection was carried out after an interval of 2 weeks with 100 μg of SpHyastatin mixed with incomplete form Freund adjuvant. Subsequently, the booster immunizations were given in the same way each week for three times in all. The blood of mice was collected 6 days after the last boost and clotted overnight at 4°C, then centrifuged at 6000 g for 10 min at 4°C to obtain the antiserum. The crude serum obtained was purified using a Nab Spin Kit, 1 mL for Antibody Purification (Thermo Scientific, USA) following the manufacturer’s protocol. The titer, purity and specificity of the purified anti-SpHyastatin antibody were analyzed using ELISA, SDS-PAGE and Western blot.
Nab spin kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nab Spin Kit is a laboratory equipment product designed for protein purification. It utilizes a spin column format to facilitate the capture and separation of target proteins from complex samples.
Automatically generated - may contain errors
5 protocols using nab spin kit
1
Generating Anti-SpHyastatin Antibody
2
Serum and CSF IgG Purification and Immunoblotting
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It was performed in a subset of 21 patients (RRMS- 16, SPMS -5). Serum and corresponding CSF IgG of each patient was initially purified (Nab Spin Kit, Thermo Fisher Scientific). Immunoblotting was done using previously published protocol.[16 (link)]
3
Affinity Purification of Rabbit Antibodies
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Rabbit polyclonal antibodies raised against purified rBap_B protein were supplied by Abyntek Biopharma S.L. (Spain). Antibodies were subsequently immunoabsorbed and purified using NAb Spin Kit (Thermoscientific).
4
Purification and Characterization of mAb
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The selected ATAC-0025 mAb was purified from concentrated supernatants using the NAb spin kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The resulting purified antibody was subsequently concentrated by means of Pierce Protein Concentrators, PES (Thermo Fisher Scientific), to obtain a concentration 0.5 mg/mL. The mAb class was determined with a commercial mouse isotyping kit MMT1 (Bio-Rad, Hercules, CA, USA). The different Ig isotype classes and light chains were determined through immune-chromatography.
5
Production and Purification of TβRII Variants
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Human 293T cells were transduced either with Lv.TβRII-SE/Fc or Lv.TβRII-Fc at a MOI of 70, in the presence of 8 μg/ml polybrene (Millipore Sigma, Burlington, MA).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted March 30, 2021. ; https://doi.org/10.1101/2021.03.01.433173 doi: bioRxiv preprint Forty-eight hours after transduction, cells were harvested, washed in phosphatebuffered saline (PBS) supplemented with 10% FBS, and the percentage of eGFP positive cells was determined by flow cytometry in a FACSCalibur device (BD Biosciences, San Jose, CA).
For the production of TβRII-SE/Fc and TβRII-Fc, lentivirally transduced 293T cells were cultured for 48 h in serum-free DMEM supplemented with Protease Inhibitor Cocktail (1/800) (Millipore Sigma, Burlington, MA). Subsequently, the conditioned media was clarified by centrifugation at 3500 rpm and filtrated through 0.22 μm filters. Recombinant protein was concentrated by centrifugation in Amicon® Ultra-15-30K (Millipore Sigma, Burlington, MA), purified on protein A/G columns (NAb™ Spin Kit, Thermo Scientific, Rockford, IL) following instructions of the manufacturer, and stored at -80°C.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted March 30, 2021. ; https://doi.org/10.1101/2021.03.01.433173 doi: bioRxiv preprint Forty-eight hours after transduction, cells were harvested, washed in phosphatebuffered saline (PBS) supplemented with 10% FBS, and the percentage of eGFP positive cells was determined by flow cytometry in a FACSCalibur device (BD Biosciences, San Jose, CA).
For the production of TβRII-SE/Fc and TβRII-Fc, lentivirally transduced 293T cells were cultured for 48 h in serum-free DMEM supplemented with Protease Inhibitor Cocktail (1/800) (Millipore Sigma, Burlington, MA). Subsequently, the conditioned media was clarified by centrifugation at 3500 rpm and filtrated through 0.22 μm filters. Recombinant protein was concentrated by centrifugation in Amicon® Ultra-15-30K (Millipore Sigma, Burlington, MA), purified on protein A/G columns (NAb™ Spin Kit, Thermo Scientific, Rockford, IL) following instructions of the manufacturer, and stored at -80°C.
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