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Sephadex protein a g beads

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Sephadex protein A/G beads are a type of chromatography resin used for the purification of antibodies. They consist of agarose-based beads that have been functionalized with either protein A or protein G, which are bacterial proteins that bind to the Fc region of immunoglobulins. The beads can be used to selectively capture and purify antibodies from complex mixtures, such as cell culture supernatants or serum samples.

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Lab products found in correlation

Nap 5 column, by GE Healthcare (4 mentions) Microbeta trilux liquid scintillation plate reader, by PerkinElmer (3 mentions) Anti myc positive control antibody, by Cell Signaling Technology (3 mentions) Rc221074, by OriGene (2 mentions) 96 well filtration plates, by Corning (2 mentions) Microbeta trilux, by PerkinElmer (2 mentions) Neg709a, by PerkinElmer (1 mentions)

Spelling variants of this product

Protein G beads (76 citations) Protein A beads (62 citations) Protein A/G (17 citations)

6 protocols using sephadex protein a g beads

1

In Vitro Protein Expression and Immunoprecipitation

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DNA plasmids containing full-length cDNA under the control of a T7 promoter for each of the validated antigens (Supplementary file 4) were verified by Sanger sequencing and used as DNA templates in the T7 TNT in vitro transcription/translation kit (Promega, Madison, WI; #L1170) using [35S]-methionine (PerkinElmer, Waltham, MA; #NEG709A). Protein was column-purified on Nap-5 columns (GE healthcare, Chicago, IL; #17-0853-01) and immunoprecipitated on Sephadex protein A/G beads (Sigma Aldrich, St. Louis, MO; #GE17-5280-02 and #GE17-0618-05, 4:1 ratio) in duplicate with serum or control antibodies in 96-well polyvinylidene difluoride filtration plates (Corning, Corning, NY; #EK-680860). Each well contained 35’000 counts per minute (cpm) of radiolabeled protein and 2.5 ul of serum or appropriately diluted control antibody (Supplementary file 4). The cpms of immunoprecipitated protein was quantified using a 96-well Microbeta Trilux liquid scintillation plate reader (Perkin Elmer).
Vazquez S.E., Ferré E.M., Scheel D.W., Sunshine S., Miao B., Mandel-Brehm C., Quandt Z., Chan A.Y., Cheng M., German M., Lionakis M., DeRisi J.L, & Anderson M.S. (2020). Identification of novel, clinically correlated autoantigens in the monogenic autoimmune syndrome APS1 by proteome-wide PhIP-Seq. eLife, 9, e55053.
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2

Radioligand Binding Assay for ZSCAN1

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The radioligand binding assay (RLBA) was performed as described previously.43 Briefly, full‐length human ZSCAN1 was in vitro transcribed and translated with [35S]‐methionine in a T7 dependent system using a ZSCAN1 plasmid containing a T7 promoter (Origene Cat: RC221074). The radiolabeled ZSCAN1 protein was then column purified and immunoprecipitated with 2.5 μL of serum or CSF per well using Sephadex protein A/G beads (Sigma Aldrich, St. Louis, MO; #GE17‐5280‐02 and #GE17‐0618‐05) in 96‐well filtration plates (Corning, Corning, NY; #EK‐680860). The plates were read out as counts per minute (cpm) using the Microbeta Trilux liquid scintillation plate reader (Perkin Elmer).
Mandel‐Brehm C., Benson L.A., Tran B., Kung A.F., Mann S.A., Vazquez S.E., Retallack H., Sample H.A., Zorn K.C., Khan L.M., Kerr L.M., McAlpine P.L., Zhang L., McCarthy F., Elias J.E., Katwa U., Astley C.M., Tomko S., Dalmau J., Seeley W.W., Pleasure S.J., Wilson M.R., Gorman M.P, & DeRisi J.L. (2022). ZSCAN1 Autoantibodies Are Associated with Pediatric Paraneoplastic ROHHAD. Annals of Neurology, 92(2), 279-291.
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3

IFN-α2 Protein Immunoprecipitation Assay

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A DNA plasmid containing full-length cDNA sequence with a Flag-Myc tag (OriGene, #RC221091) was verified by Sanger sequencing and used as template in T7-promoter–based in vitro transcription/translation reactions (Promega, #L1170) using [S35]-methionine (PerkinElmer, #NEG709A). IFN-α2 protein was column-purified using NAP-5 columns (GE Healthcare, #17-0853-01); incubated with 2.5 μl of serum, 2.5 μl of plasma, or 1 μl of anti-myc–positive control antibody (Cell Signaling Technology, #2272); and immunoprecipitated with Sephadex protein A/G beads (4:1 ratio; Sigma-Aldrich, #GE17-5280-02 and #GE17-0618-05) in 96-well polyvinylidene difluoride filtration plates (Corning, #EK-680860). The radioactive counts [counts per minute (cpm)] of immunoprecipitated protein were quantified using a 96-well MicroBeta TriLux liquid scintillation plate reader (PerkinElmer). Antibody index for each sample was calculated as follows: (sample cpm value – mean blank cpm value)/(positive control antibody cpm value – mean blank cpm value). For the COVID-19 patient and CCP cohorts, a positive signal was defined as greater than 6 standards deviations above the mean of pre–COVID-19 blood bank non-inflammatory controls.
Bastard P., Vazquez S., Liu J., Laurie M.T., Wang C.Y., Gervais A., Le Voyer T., Bizien L., Zamecnik C., Philippot Q., Rosain J., Catherinot E., Willmore A., Mitchell A.M., Bair R., Garçon P., Kenney H., Fekkar A., Salagianni M., Poulakou G., Siouti E., Sahanic S., Tancevski I., Weiss G., Nagl L., Manry J., Duvlis S., Arroyo-Sánchez D., Paz Artal E., Rubio L., Perani C., Bezzi M., Sottini A., Quaresima V., Roussel L., Vinh D.C., Reyes L.F., Garzaro M., Hatipoglu N., Boutboul D., Tandjaoui-Lambiotte Y., Borghesi A., Aliberti A., Cassaniti I., Venet F., Monneret G., Halwani R., Sharif-Askari N.S., Danielson J., Burrel S., Morbieu C., Stepanovskyy Y., Bondarenko A., Volokha A., Boyarchuk O., Gagro A., Neuville M., Neven B., Keles S., Hernu R., Bal A., Novelli A., Novelli G., Saker K., Ailioaie O., Antolí A., Jeziorski E., Rocamora-Blanch G., Teixeira C., Delaunay C., Lhuillier M., Le Turnier P., Zhang Y., Mahevas M., Pan-Hammarström Q., Abolhassani H., Bompoil T., Dorgham K., Gorochov G., Laouenan C., Rodríguez-Gallego C., Ng L.F., Renia L., Pujol A., Belot A., Raffi F., Allende L.M., Martinez-Picado J., Ozcelik T., Keles S., Imberti L., Notarangelo L.D., Troya J., Solanich X., Zhang S.Y., Puel A., Wilson M.R., Trouillet-Assant S., Abel L., Jouanguy E., Ye C.J., Cobat A., Thompson L.M., Andreakos E., Zhang Q., Anderson M.S., Casanova J.L, & DeRisi J.L. (2022). Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs. Science Immunology.
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4

Detecting SARS-CoV-2 Antibodies via Immunoprecipitation

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A DNA plasmid containing full-length cDNA sequence with a Flag-Myc tag (OriGene, #RC221091) was verified by Sanger sequencing and used as template in T7-promoter–based in vitro transcription/translation reactions (Promega, #L1170) using [S35]-methionine (PerkinElmer, #NEG709A). IFN-α2 protein was column-purified using NAP-5 columns (GE Healthcare, #17-0853-01); incubated with 2.5 μl of serum, 2.5 μl of plasma, or 1 μl of anti-myc–positive control antibody (Cell Signaling Technology, #2272); and immunoprecipitated with Sephadex protein A/G beads (4:1 ratio; Sigma-Aldrich, #GE17-5280-02 and #GE17-0618-05) in 96-well polyvinylidene difluoride filtration plates (Corning, #EK-680860). The radioactive counts [counts per minute (cpm)] of immunoprecipitated protein were quantified using a 96-well MicroBeta TriLux liquid scintillation plate reader (PerkinElmer). Antibody index for each sample was calculated as follows: (sample cpm value – mean blank cpm value)/(positive control antibody cpm value – mean blank cpm value). For the COVID-19 patient and CCP cohorts, a positive signal was defined as greater than 6 SDs above the mean of pre–COVID-19 blood bank noninflammatory controls. For the large asymptomatic San Francisco community population cohort, a positive signal was defined as having a z score greater than 3.3 (P = 0.0005) relative to the whole cohort.
van der Wijst M.G., Vazquez S.E., Hartoularos G.C., Bastard P., Grant T., Bueno R., Lee D.S., Greenland J.R., Sun Y., Perez R., Ogorodnikov A., Ward A., Mann S.A., Lynch K.L., Yun C., Havlir D.V., Chamie G., Marquez C., Greenhouse B., Lionakis M.S., Norris P.J., Dumont L.J., Kelly K., Zhang P., Zhang Q., Gervais A., Le Voyer T., Whatley A., Si Y., Byrne A., Combes A.J., Rao A.A., Song Y.S., Fragiadakis G.K., Kangelaris K., Calfee C.S., Erle D.J., Hendrickson C., Krummel M.F., Woodruff P.G., Langelier C.R., Casanova J.L., Derisi J.L., Anderson M.S, & Ye C.J. (2021). Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19. Science Translational Medicine, 13(612), eabh2624.
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5

IFN-α2 Autoantibody Immunoprecipitation Assay

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A DNA plasmid containing full-length cDNA sequence with a Flag-Myc tag (Origene #RC221091) was verified by Sanger sequencing and used as template in T7-promoter-based in vitro transcription/translation reactions (Promega, #L1170) using [S35]-methionine (Perkin Elmer, #NEG709A). IFN-α2 protein was column-purified using Nap-5 columns (GE Healthcare, #17-0853-01), incubated with 2.5μl serum, 2.5μl plasma, or 1μl anti-myc positive control antibody (Cell Signal, #2272), and immunoprecipitated with Sephadex protein A/G beads (Sigma Aldrich, #GE17-5280-02 and #GE17-0618-05, 4:1 ratio) in 96-well polyvinylidene difluoride filtration plates (Corning, #EK-680860). The radioactive counts (cpms) of immunoprecipitated protein was quantified using a 96-well Microbeta Trilux liquid scintillation plate reader (Perkin Elmer). Antibody index for each sample was calculated as follows: (sample cpm value – mean blank cpm value) / (positive control antibody cpm value – mean blank cpm value). For the COVID-19 patient and convalescent plasma cohorts, a positive signal was defined as greater than 6 standard deviations above the mean of pre-COVID-19 blood bank non-inflammatory controls. For the large asymptomatic San Francisco community population cohort, a positive signal was defined as having a z-score greater than 3.3 (p=0.0005) relative to the whole cohort.
van der Wijst M.G., Vazquez S.E., Hartoularos G.C., Bastard P., Grant T., Bueno R., Lee D.S., Greenland J.R., Sun Y., Perez R., Ogorodnikov A., Ward A., Mann S.A., Lynch K.L., Yun C., Havlir D.V., Chamie G., Marquez C., Greenhouse B., Lionakis M.S., Norris P.J., Dumont L.J., Kelly K., Zhang P., Zhang Q., Gervais A., Le Voyer T., Whatley A., Si Y., Byrne A., Combes A.J., Rao A.A., Song Y.S., Fragiadakis G.K., Kangelaris K., Calfee C.S., Erle D.J., Hendrickson C., Krummel M.F., Woodruff P.G., Langelier C.R., Casanova J.L., Derisi J.L., Anderson M.S, & Ye C.J. (2021). Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19. Science Translational Medicine, 13(612), eabh2624.
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6

ZSCAN1 In Vitro Binding Assay

Cited 1 time
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The RLBA was performed as described previously43 (link). Briefly, full-length human ZSCAN1 was in vitro transcribed and translated with [35S]-methionine in a T7 dependent system using a ZSCAN1 plasmid containing a T7 promoter (Origene Cat: RC221074). The radiolabeled ZSCAN1 protein was then column purified and immunoprecipitated with 2.5ul serum or CSF per well using Sephadex protein A/G beads (Sigma Aldrich, St. Louis, MO; #GE17–5280-02 and #GE17–0618-05) in 96-well filtration plates (Corning, Corning, NY; #EK-680860). The plates were read out as counts per minute (cpm) using the Microbeta Trilux liquid scintillation plate reader (Perkin Elmer).
Mandel‐Brehm C., Benson L.A., Tran B., Kung A.F., Mann S.A., Vazquez S.E., Retallack H., Sample H.A., Zorn K.C., Khan L.M., Kerr L.M., McAlpine P.L., Zhang L., McCarthy F., Elias J.E., Katwa U., Astley C.M., Tomko S., Dalmau J., Seeley W.W., Pleasure S.J., Wilson M.R., Gorman M.P, & DeRisi J.L. (2022). ZSCAN1 Autoantibodies Are Associated with Pediatric Paraneoplastic ROHHAD. Annals of Neurology, 92(2), 279-291.
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