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Nab protein a g spin kit

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The NAb Protein A/G Spin Kit is a laboratory product designed for the purification of antibodies from a variety of sample types. It utilizes Protein A/G affinity chromatography to selectively bind and capture antibodies, allowing for their efficient separation and recovery. The kit provides a convenient spin column format for a streamlined purification process.

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Lab products found in correlation

Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions) Alexa fluor 555 goat anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions) Dapi vectashield hard set, by Vector Laboratories (2 mentions) Te2000 e eclipse inverted fluorescent microscope, by Nikon (2 mentions) Volocity software, by PerkinElmer (2 mentions) Polyethylene glycol, by Merck Group (2 mentions) Normal control human plasma, by Hyphen Biomed (2 mentions) Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions) Phosphatidylserine ps, by Merck Group (1 mentions) Ctla 4 blockade bioassay, by Promega (1 mentions) Synergy 2 plate reader, by Agilent Technologies (1 mentions) Amicon ultra centrifugal filter, by Merck Group (1 mentions) Alexa fluor 488 antibody, by Jackson ImmunoResearch (1 mentions) Zeba spin column, by Thermo Fisher Scientific (1 mentions) Lsrfortessa x 20, by BD (1 mentions) Oligo clean concentrator kit, by Zymo Research (1 mentions) Zeba spin desalting columns0.5 ml, by Thermo Fisher Scientific (1 mentions) Liveblazer fret b g loading kit, by Thermo Fisher Scientific (1 mentions) Geneblazer fret based β lactamase reporter assay, by Thermo Fisher Scientific (1 mentions) Sense microplate reader, by Hidex (1 mentions)

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Protein A/G (47 citations) Nab Protein G Spin Kit (21 citations) Nab Spin Kit (5 citations)

18 protocols using nab protein a g spin kit

1

Serum IgG Purification from Fasted Samples

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Blood samples for the present study were obtained post-test (i.e. after the diagnostic HUT was performed) following overnight fasting. A trained nurse performed an antecubital venipuncture in a dedicated room after 10 min rest in supine position. Serum was separated by centrifugation and stored at −80°C prior to shipping of duplicate aliquots in dry ice to the laboratory in Oklahoma City. The frozen integrity of each aliquot was confirmed upon arrival and samples were immediately placed in a −80°C freezer prior to thawing of one aliquot for the first assay. Serum IgG was purified using the NAb Protein A/G Spin Kit (Pierce Biotechnology). Receipt and assay of these de-identified samples was approved by the University of Oklahoma Health Sciences Center Institutional Review Board.
Fedorowski A., Li H., Yu X., Koelsch K.A., Harris V.M., Liles C., Murphy T.A., Quadri S.M., Scofield R.H., Sutton R., Melander O, & Kem D.C. (2016). Antiadrenergic autoimmunity in postural tachycardia syndrome. Europace, 19(7), 1211-1219.
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2

Serum Immunoglobulin G Purification

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Blood samples for the present study were obtained after overnight fasting and were from the same group, as previously described.7 Serum was separated by centrifugation and stored at −80°C before shipping of duplicate aliquots in dry ice to the laboratory in Oklahoma City. The frozen integrity of each aliquot was confirmed on arrival, and samples were immediately placed in a −80°C freezer before thawing of one aliquot for the first assay. The identity of each aliquot was blinded to the laboratory personnel until after the assays were completed. Serum immunoglobulin G (IgG) was purified using the NAb Protein A/G Spin Kit (Pierce Biotechnology).
Yu X., Li H., Murphy T.A., Nuss Z., Liles J., Liles C., Aston C.E., Raj S.R., Fedorowski A, & Kem D.C. (2018). Angiotensin II Type 1 Receptor Autoantibodies in Postural Tachycardia Syndrome. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 7(8), e008351.
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3

Immunostaining of RNA:DNA Hybrids

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For staining of RNA:DNA hybrids in FA-D2 cell lines, S9.6 antibody was purified from the mouse BALB/c hybridoma cells (gift of Tae Hoon Kim, Yale University) using NAb Protein A/G Spin Kit (Thermo Fisher) according to the manufacturer’s instructions. Cells were plated in 8 chamber slides, grown to 50% confluence, and then treated with 500 nM MMC (Sigma) for the indicated time. Slides were then rinsed in PBS, fixed in 4% paraformaldehyde in PBS for 5 minutes, rinsed in PBS again, permeabilized in 0.5% Triton X-100 in PBS for 5 minutes, rinsed in PBS once more, and then blocked in PBS with 0.5% BSA, 0.1% NP-40 and 10% normal goat serum overnight at 4°C. Blocking agent was aspirated, and 1 μg/ml of S9.6 antibody in PBS with 0.1% NP-40 and 0.5% BSA was added to the slides, followed by an overnight incubation at 4°C. Slides were then washed three times in 0.1% PBS-T, and goat anti-mouse Alexa Fluor 555 secondary antibody (A-21422, Thermo Fisher) diluted 1:1000 was added to slides for 2 hours at room temperature. After washing 3 times in 0.1% PBS-T and 2 more times in PBS, slides were mounted using DAPI Vectashield Hard-Set (Vector Laboratories) and images were captured in a TE2000-E Eclipse inverted fluorescent microscope (Nikon). Volocity software (Perkin Elmer) was used to quantify immunofluorescence.
Liang Z., Liang F., Teng Y., Chen X., Liu J., Longerich S., Rao T., Green A.M., Collins N.B., Xiong Y., Lan L., Sung P, & Kupfer G.M. (2019). Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination. Cell reports, 26(3), 564-572.e5.
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4

Epitope-mimicking Peptides Modulate GnRHR-AAb Activity

Cited 2 times
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The effect of epitope-mimicking retro-inverso peptides on neutralizing GnRHR-AAb activity was examined using the GeneBLAzer GnRHR cell-based β-lactamase reporter assay (Invitrogen) as previously described [4 (link),5 (link)]. Briefly, serum IgG samples (100 μg/mL) purified using NAb Protein A/G Spin Kit (Thermo Fisher Scientific) were added to the assay plate and incubated with the cells for 5 hours in the absence and presence of an overnight preincubation with a 10-fold molar excess of the retro-inverso peptides. The β-lactamase substrate CCF4-AM was then added, and the GnRHR response was recorded. Data were expressed as fold increase over buffer baseline to normalize the individual values.
Li H., Guo Y., Deng J., Gali H., Weedin E.A., Burks H.R., Craig L.B, & Yu X. (2021). GnRH receptor-activating autoantibodies in polycystic ovary syndrome: identification of functional epitopes and development of epitope mimetic inhibitors. Endocrine, 75(3), 959-963.
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5

Development of Anti-EV71 3Dpol mAbs

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IgG mAbs against EV71 3Dpol were developed by traditional hybridoma technique as previously described [23 (link)]. In brief, 5-wk-old female SPF BALB/c mice were immunized subcutaneously with 100 μg of 3Dpol at 2-wk interval. Four wks after the last booster and 3 days before cell fusion, the mice were boosted with 200 μg of 3Dpol. Three days later, murine splenocytes were harvested and fused with SP2/0 using 50% polyethyleneglycol (Sigma-Aldrich, MO, USA). Hybridoma culture supernatants were preliminarily screened by EV71 3Dpol protein using ELISA. The positive hybridoma cells were cloned by a limiting dilution and the stable hybridoma clones were injected into liquid paraffin-pretreated abdominal cavities of BALB/c mice. Subsequently, the mAbs were harvested and purified from the ascite with an antibody purification kit according to the manufacturer’s specifications (NAb™ Protein A/G Spin Kit, Thermo Scientific, IL, USA). This mouse study was approved by the ethics committee of life science and research in Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS) (No. WIVA09201502).
Li Y., Yu J., Qi X, & Yan H. (2019). Monoclonal antibody against EV71 3Dpol inhibits the polymerase activity of RdRp and virus replication. BMC Immunology, 20, 6.
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6

Phospholipid Binding Assay for IgG, IgM, and IgA

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All of the phospholipids used in this paper were purchased from Avanti polar lipids, which included 16:0 PE (cat# 850705X); 18:0 PE (cat# 850715X); 18:1 PE (cat# 850725C), 20:4 PE (cat# 850800C); 22:6 PE (cat# 850797C); and egg PE (cat# 840021C).
Pooled normal human sera, BSA and phosphatidylserine (PS) were purchased from Sigma-Aldrich. ABP was a kind of gift from Dr. Dawn R. Wagenknecht. Both BSA and ABP were dissolved in 1X PBS and filtered with a 0.22 micron syringe filter before use. Anti-human IgG (Cat# A9544), IgM (Cat# A3275) and IgA (cat# A9669) alkaline phosphatase and p-nitrophenyl phosphate liquid substrate system (cat# N7656) were obtained from Sigma-Aldrich. NAb™ protein A/G spin kit (Cat# 89950) was obtained from Thermo Fisher Scientific.
Hou S., Harper P.E., Bardin N, & Zhao M. (2016). The outcome of ELISA for antiphosphatidylethanolamine antibodies is dependent on the composition of phosphatidylethanolamine. Journal of immunological methods, 440, 27-34.
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7

IgA Purification and Transfer Protocol

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Small intestines were removed from naïve WT and Igha−/− mice, cut open longitudinally and mucus was scrapped out and suspended in PBS. Luminal content was filtered and spun twice at 8000g for 5 minutes, the bacterial pellets discarded, and the supernatant collected was further processed on a Nab Protein A/G Spin kit (Thermo Fisher Scientific) according to the manufacturer’s instruction. The concentration of IgA in the eluted fraction, depleted of IgG, was then measured by ELISA (Bethyl). Igha−/− recipient mice received by oral gavage 100μg of purified IgA the day before and day 3 after LCWE i.p. injection.
Rivas M.N., Wakita D., Franklin M.K., Carvalho T.T., Abolhesn A., Gomez A.C., Chen S., Lehman T.J., Sato K., Shibuya A., Fasano A., Kiyono H., Abe M., Tatsumoto N., Yamashita M., Crother T.R., Shimada K, & Arditi M. (2019). Intestinal permeability and IgA provoke immune vasculitis linked to cardiovascular inflammation. Immunity, 51(3), 508-521.e6.
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8

Generation of Ebola Virus-Specific Monoclonal Antibodies

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The preparation of Ebola virus GP-specific mAbs were generated as previously described [14 (link)]. In brief, 5-week-old female BALB/c mice 6–8 weeks old were immunized 50 μg rZEBOV GPdTM with 3-weeks’ interval. At four weeks after the last booster and 3 days before cell fusion, the mice were boosted with 200 μg of the rZEBOV GPdTM. Three days later, mice splenocytes were harvested and fused with SP2/0 using 50% polyethyleneglycol (Sigma-Aldrich, MO). Hybridoma was screened using indirect ELISA. The positive hybridoma cells were cloned by a limiting dilution and the stable hybridoma clones were injected into liquid paraffin-pretreated abdominal cavities of BALB/c mice. Subsequently, the mAbs were harvested and purified from the seroperitoneum with an antibody purification kit according to the manufacturer’s specifications (NAb™ Protein A/G Spin Kit, Thermo Scientific, USA).
Zai J., Yi K., Xie L., Zhu J., Feng X, & Li Y. (2018). Dual monoclonal antibody-based sandwich ELISA for detection of in vitro packaged Ebola virus. Diagnostic Pathology, 13, 96.
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9

CTLA-4 Blockade Bioassay for T Cell Activation

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T cell activation after CTLA-4 blockade was assessed using the CTLA-4 Blockade Bioassay (Promega), according to manufacturer’s instructions. Ipilimumab and tremelimumab DMAb was purified from individual mice for this assay (n=3 mice for each DMAb), using the Nab Protein A/G Spin Kit (ThermoFisher), and was concentrated using Amicon Ultra Centrifugal Filters (Millipore Sigma). Luciferase activity was measured using the Synergy2 plate reader (Biotek).
Duperret E.K., Trautz A., Stoltz R., Patel A., Wise M.C., Perales-Puchalt A., Smith T., Broderick K.E., Masteller E., Kim J.J., Humeau L., Muthumani K, & Weiner D.B. (2018). Synthetic DNA-encoded monoclonal antibody delivery of anti-CTLA-4 antibodies induces tumor shrinkage in vivo. Cancer research, 78(22), 6363-6370.
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10

CHOV-GP Specific IgG Antibody Assay

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Serum specific IgG antibodies against CHOV-GP were detected by a cell-based assay and flow cytometry. Briefly, CHOV GPs-293T were used to detect binding of purified IgGs (10 µg/ml for IgG determination) from subjects (15 acute and 15 convalescent, 33 seropositive and 14 healthy donors). Polyclonal IgG was isolated from serum using NAb™ Protein A/G Spin Kit (Thermo Scientific) followed by desalting using Zeba spin columns (Thermo Scientific) according to manufacturer´s instructions.
Reactive cells were detected with Alexa Fluor 488 anti-human IgG antibody (Jackson Immuno Research). For IgG subclass determination, CHOV GPs-293T cells were incubated with sera (1/1000), washed, stained with anti-human -IgG1, -IgG2, -IgG3, or -IgG4 mouse antibody (Southern Biotech) and labeled with anti-mouse Alexa Fluor 488 antibody (Jackson ImmunoResearch). All antibody stainings were incubated for 1 hour at 4°C. The levels of CHOV GP-specific antibody binding were analyzed by flow cytometry (BD LSRFortessa X-20).
Salinas T.P., Garrido J.L., Salazar J.R., Gonzalez P., Zambrano N., Fuentes-Villalobos F., Bravo F., Fica-Leon V., Salas-Burgos A., Calvo M., Alvarez R., Armien B, & Barria M.I. (2021). Cytokine Profiles and Antibody Response Associated to Choclo Orthohantavirus Infection. Frontiers in Immunology, 12, 603228.
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