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Nab protein a plus spin kit

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The NAb Protein A Plus Spin Kit is a laboratory equipment used for the purification of antibodies from cell culture supernatants or ascites fluid. It utilizes Protein A resin in a spin column format to capture and isolate immunoglobulins.

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Lab products found in correlation

Pei max, by Polysciences (4 mentions) Pd 10 gel filtration column, by GE Healthcare (1 mentions) Dmem low glucose, by Fujifilm (1 mentions) Goat anti rabbit igg h l hrp, by Abcam (1 mentions) Donkey anti sheep igg hrp, by Merck Group (1 mentions) Anti lc3, by Novus Biologicals (1 mentions) Anti ubiquitin p4d1, by Cell Signaling Technology (1 mentions) Anti ulk1, by Cell Signaling Technology (1 mentions) Anti lc3, by Cell Signaling Technology (1 mentions) Anti gabarap, by Abcam (1 mentions) Anti myc, by Santa Cruz Biotechnology (1 mentions) Anti pulk1 ser555, by Cell Signaling Technology (1 mentions) Anti usp5, by Proteintech (1 mentions) Anti ref 2 p, by Abcam (1 mentions) Anti tubulin, by Merck Group (1 mentions) Anti ha, by Merck Group (1 mentions) Anti flag m2, by Merck Group (1 mentions) Anti gfp, by Abcam (1 mentions) Pathhunter express β arrestin gpcr assay kit, by Eurofins (1 mentions) Bca protein assay, by Solarbio (1 mentions)

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Nab Spin Kit (5 citations) PLUSTM (3 citations)

10 protocols using nab protein a plus spin kit

1

Heptapeptide Hapten Conjugation to KLH

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The heptapeptide was synthesized on solid phase using standard Fmoc main chain and Boc/Trt side chain protection chemistry. The VHCSAG peptide was treated with bromo-dimedone to generate the VHCDMDSAG modified peptide. KLH (Keyhole Limpet Hemocyanin) protein was activated by adding sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylated), a hetero-bifunctional crosslinker containing an amine-reactive N-hydroxysuccininmide and a sulfhydryl-reactive malemide group. The hapten was conjugated to succinylated KLH overnight at pH 8 and the product was then purified over a PD10 gel filtration column (GE Healthcare). Two New Zealand White (specific pathogen-free grade) rabbits were immunized subcutaneiously with conjugate in a 50:50 emulsification with adjuvant [either Freunds complete (FCA) or Freunds incomplete (FIA) according to the following schedule: day 0 boost (FCA), day 14 boost (FIA), and day 28 boost (FIA). The rabbits were bled on days 35 and 40 after the primary boost, the red blood cells spun out by centrifugation, and the remaining antisera was IgG purified using a Nab Protein A Plus Spin Kit (Thermo Scientific).
Garcia F.J, & Carroll K.S. (2016). An Immunochemical Approach to Detect Oxidized Protein Tyrosine Phosphatases Using a Selective C-Nucleophile Tag. Molecular bioSystems, 12(6), 1790-1798.
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2

Monoclonal Antibody Production Protocol

Cited 1 time
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Ten monoclonal antibodies (adintrevimab, bamlanivimab, bebtelovimab, casirivimab, cilgavimab, etesevimab, imdevimab, regdanvimab, sotrovimab and tixagevimab) were prepared as previously described (Meng et al., 2022 (link); Yamasoba et al., 2022b (link); Liu et al., 2021 (link)). Briefly, the pCAGGS vectors containing the sequences encoding the immunoglobulin heavy and light chains were cotransfected into HEK293T cells at a 1:1 ratio using PEI Max (Polysciences, Cat# 24765-1). The culture medium was refreshed with DMEM (low glucose) (Wako, Cat# 041-29775) containing 10% FBS without PS. At 72 hours posttransfection, the culture medium was harvested, and the antibodies were purified using an NAb protein A plus spin kit (Thermo Fisher Scientific, Cat# 89948) according to the manufacturer’s protocol.
Saito A., Tamura T., Zahradnik J., Deguchi S., Tabata K., Anraku Y., Kimura I., Ito J., Yamasoba D., Nasser H., Toyoda M., Nagata K., Uriu K., Kosugi Y., Fujita S., Shofa M., Monira Begum M., Shimizu R., Oda Y., Suzuki R., Ito H., Nao N., Wang L., Tsuda M., Yoshimatsu K., Kuramochi J., Kita S., Sasaki-Tabata K., Fukuhara H., Maenaka K., Yamamoto Y., Nagamoto T., Asakura H., Nagashima M., Sadamasu K., Yoshimura K., Ueno T., Schreiber G., Takaori-Kondo A., Shirakawa K., Sawa H., Irie T., Hashiguchi T., Takayama K., Matsuno K., Tanaka S., Ikeda T., Fukuhara T, & Sato K. (2022). Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant. Cell Host & Microbe, 30(11), 1540-1555.e15.
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3

Purification and Characterization of Polyclonal BTV-rVP2 Antibodies

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Polyclonal rabbit antisera, raised against individual BTV-rVP2 proteins, were purified using the NAb Protein A Plus Spin Kit (ThermoFisher Scientific; Waltham, MA, USA) as per manufacturer’s instructions. Antibody concentration (mg/mL) was determined by spectrophotometer (Eppendorf; Stevenage, UK) at an absorbance of 280 nm. Secondary antibodies were obtained from commercial suppliers, diluted and used as follows: goat anti-rabbit IgG H&L (HRP; Abcam, Cambridge, UK), 1:2000; donkey anti sheep IgG (HRP; Sigma Aldrich, Gillingham, UK), 1:5000.
Fay P.C., Mohd Jaafar F., Batten C., Attoui H., Saunders K., Lomonossoff G.P., Reid E., Horton D., Maan S., Haig D., Daly J.M, & Mertens P.P. (2021). Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes. Viruses, 13(8), 1455.
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4

Antibodies for Autophagy Protein Analysis

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Antibodies used for this study were anti-LC3 (Cell Signaling, 4108), anti-LC3 (Novus, NB100-2220), anti-Ref(2)P (Abcam, ab178440), anti-GABARAP (Abcam, ab109364), anti-pULK1-Ser555 (Cell signaling, 5869), anti-ULK1 (Cell signaling, 8054), anti-USP5 (Proteintech, 10473-1-AP), anti-p62 (MBL, PM045), anti-Flag M2 (Sigma, F1804), anti-MYC (Santa Cruz, SC-40), anti-HA (Sigma, H9658), anti-ACTB/β-actin (Novus, NB600-501), anti-tubulin (Sigma, T0198), anti-GFP (Abcam, ab290), anti-ubiquitin (FK2; Sigma, ST1200), and anti-ubiquitin (P4D1; Cell signaling, 3936). To generate antiserum against Drosophila Atg1, the C-terminal segment of Atg1 (amino acid residues 562-855) was cloned to pET32a vector to generate 6xHis-tagged fusion protein. The fusion protein was purified using a Ni Sephsrose 6 FF column (GE Healthcare). The purified protein was used to immunize rabbits by LTK BioLaboratories and the resulting antiserum was purified by NAb Protein A Plus Spin Kit (Thermo).
Pai Y.L., Lin Y.J., Peng W.H., Huang L.T., Chou H.Y., Wang C.H., Chien C.T, & Chen G.C. (2023). The deubiquitinase Leon/USP5 interacts with Atg1/ULK1 and antagonizes autophagy. Cell Death & Disease, 14(8), 540.
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5

Recombinant Anti-SARS-CoV-2 Antibody Production

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Casirivimab and Imdevimab were prepared as previously described54 . To construct the plasmids expressing anti-SARS-CoV-2 monoclonal antibodies (Casirivimab and Imdevimab), the sequences of the variable regions of Casirivimab and Imdevimab were obtained from KEGG Drug Database (https://www.genome.jp/kegg/drug/) and were artificially synthesized (Fasmac). The obtained coding sequences of the variable regions of the heavy and light chains were cloned into the pCAGGS vector containing the sequences of the human immunoglobulin 1 and kappa constant region [kindly provided by Dr. Hisashi Arase (Osaka University, Japan)].
To prepare these monoclonal antibodies, the pCAGGS vectors containing the sequences encoding the immunoglobulin heavy and light chains were cotransfected into HEK293T cells using PEI Max (Polysciences, Cat# 24765–1). At 48 h posttransfection, the cell culture supernatants were harvested, and the antibodies were purified using NAb protein A plus spin kit (Thermo Fisher Scientific, Cat# 89948) according to the manufacturer’s protocol.
, & Gupta R. (2022). SARS-CoV-2 Omicron spike mediated immune escape and tropism shift. Research Square.
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6

Purification and Application of IgGs

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Samples of serum from patients or control donors were applied to minicolumns of protein A Sepharose (Nab Protein A Plus Spin Kit, Thermofisher) according to the manufacturer’s instructions. Eluates were then concentrated using the Protein Concentrators kit (ThermoFisher). Purified IgGs were then used at a physiological concentration that is expected in human serum (12-14 g/L). Equal volumes of the patients’ purified IgGs or the nonretained fractions were added to control cPRP.
Lee C.S., Huguenin Y., Pillois X., Moulieras M., Marcy E., Whittaker S., Chen V.M, & Fiore M. (2023). In vitro characterization of rare anti-αIIbβ3 isoantibodies produced by patients with Glanzmann thrombasthenia that severely block fibrinogen binding and generate procoagulant platelets via complement activation. Research and Practice in Thrombosis and Haemostasis, 8(1), 102253.
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7

Production of Anti-SARS-CoV-2 Monoclonal Antibodies

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Casirivimab and Imdevimab were prepared as previously described53 (link). To construct the plasmids expressing anti-SARS-CoV-2 monoclonal antibodies (casirivimab and imdevimab), the sequences of the variable regions of casirivimab and imdevimab were obtained from KEGG Drug Database (https://www.genome.jp/kegg/drug/) and were artificially synthesized (Fasmac). The obtained coding sequences of the variable regions of the heavy and light chains were cloned into the pCAGGS vector containing the sequences of the human immunoglobulin 1 and κ constant region (provided by H. Arase).
To prepare these monoclonal antibodies, the pCAGGS vectors containing the sequences encoding the immunoglobulin heavy and light chains were cotransfected into HEK293T cells using PEI Max (Polysciences, 24765-1). At 48 h after transfection, the cell culture supernatants were collected, and the antibodies were purified using the NAb protein A plus spin kit (Thermo Fisher Scientific, 89948) according to the manufacturer’s protocol.
Meng B., Abdullahi A., Ferreira I.A., Goonawardane N., Saito A., Kimura I., Yamasoba D., Gerber P.P., Fatihi S., Rathore S., Zepeda S.K., Papa G., Kemp S.A., Ikeda T., Toyoda M., Tan T.S., Kuramochi J., Mitsunaga S., Ueno T., Shirakawa K., Takaori-Kondo A., Brevini T., Mallery D.L., Charles O.J., Bowen J.E., Joshi A., Walls A.C., Jackson L., Martin D., Smith K.G., Bradley J., Briggs J.A., Choi J., Madissoon E., Meyer K.B., Mlcochova P., Ceron-Gutierrez L., Doffinger R., Teichmann S.A., Fisher A.J., Pizzuto M.S., de Marco A., Corti D., Hosmillo M., Lee J.H., James L.C., Thukral L., Veesler D., Sigal A., Sampaziotis F., Goodfellow I.G., Matheson N.J., Sato K, & Gupta R.K. (2022). Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity. Nature, 603(7902), 706-714.
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8

Detecting M2R Autoantibodies and Allosteric Effects

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Antibodies produced in the rabbit sera were detected by ELISA as described [15 (link)]. Briefly, ELISA plates were coated with the M2R ECL2 peptide at 10 μg/mL. Sera were diluted 1:10,000, and goat anti-rabbit IgG conjugated with alkaline phosphatase and its substrate paranitrophenyl phosphate 104 were used to detect antibody binding. The optical density (OD) values were read at 405 nm at 60 min.
Rabbit M2R-AAb activity was assessed by serum IgG-induced M2R activation in M2R-transfected cells using the PathHunter eXpress β-arrestin GPCR assay kit (DiscoveRx, Fremont, CA) as described [28 (link)]. Briefly, cells cultured on a 96-well plate were incubated with rabbit IgG (0.1 μg/mL) purified from the pre- and postimmune sera using the NAb™ Protein A Plus Spin Kit (Thermo Fisher Scientific), followed by incubation with PathHunter detection reagents. Inhibition of rabbit IgG-induced M2R activation was tested with the muscarinic blocker atropine (10 μmol/L). The luminescence signal was read on a luminometer, and the β-arrestin recruitment levels are expressed as relative luminescence unit (RLU).
Dosage response curves for APE (10−9-10−5 mol/L) in the absence and presence of rabbit serum IgG (0.1 μg/mL) were generated to examine the allosteric effect of M2R-AAb on APE-induced M2R activation in vitro.
Deng J., Li H., Guo Y., Zhang G., Fischer H., Stavrakis S, & Yu X. (2022). Transcutaneous vagus nerve stimulation attenuates autoantibody-mediated cardiovagal dysfunction and inflammation in a rabbit model of postural tachycardia syndrome. Journal of interventional cardiac electrophysiology : an international journal of arrhythmias and pacing, 66(2), 291-300.
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9

Purification and Validation of IgG from APS Sera

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IgG was purified from APS or control sera with a NAb™ Protein A Plus Spin Kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer's instructions and as previously described.24 Briefly, sera were passed through a Protein A Agarose Column at least three times. IgG was then eluted with 0.1 mol/L glycine and neutralized with 1 mol/L Tris. IgG purified from APS sera was termed APS‐IgG. IgG purified from control sera was termed HC‐IgG. IgG concentrations were determined by a BCA protein assay (Solarbio, Beijing, China) according to manufacturer's instructions. IgG purity was verified with Coomassie staining. All IgG samples were determined to contain no detectable endotoxins using a Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific) according to manufacturer's instructions.
Lu Y., Dong Y., Zhang Y., Shen D., Wang X., Ge R., Zhang M., Xia Y, & Wang X. (2020). Antiphospholipid antibody‐activated NETs exacerbate trophoblast and endothelial cell injury in obstetric antiphospholipid syndrome. Journal of Cellular and Molecular Medicine, 24(12), 6690-6703.
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10

Antibody Production and Purification

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Casirivimab, imdevimab and sotrovimab were prepared as previously described (Liu et al., 2021b (link); Meng et al., 2022 (link)). Briefly, the pCAGGS vectors containing the sequences encoding the immunoglobulin heavy and light chains were cotransfected into HEK293T cells using PEI Max (Polysciences, Cat# 24765-1). At 48 h posttransfection, the cell culture supernatants were harvested, and the antibodies were purified using NAb protein A plus spin kit (Thermo Fisher Scientific, Cat# 89948) according to the manufacturer’s protocol.
Yamasoba D., Kimura I., Nasser H., Morioka Y., Nao N., Ito J., Uriu K., Tsuda M., Zahradnik J., Shirakawa K., Suzuki R., Kishimoto M., Kosugi Y., Kobiyama K., Hara T., Toyoda M., Tanaka Y.L., Butlertanaka E.P., Shimizu R., Ito H., Wang L., Oda Y., Orba Y., Sasaki M., Nagata K., Yoshimatsu K., Asakura H., Nagashima M., Sadamasu K., Yoshimura K., Kuramochi J., Seki M., Fujiki R., Kaneda A., Shimada T., Nakada T.A., Sakao S., Suzuki T., Ueno T., Takaori-Kondo A., Ishii K.J., Schreiber G., Sawa H., Saito A., Irie T., Tanaka S., Matsuno K., Fukuhara T., Ikeda T, & Sato K. (2022). Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike. Cell, 185(12), 2103-2115.e19.
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