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Ni nta hissorb 96 well plates

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Ni-NTA HisSorb 96-well plates are a type of laboratory equipment used for the efficient purification of histidine-tagged recombinant proteins. The plates are coated with nickel-nitrilotriacetic acid (Ni-NTA) resin, which selectively binds to the histidine tag on the target protein, allowing for easy separation and purification.

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Lab products found in correlation

3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (2 mentions) Prism 5, by GraphPad (2 mentions) S1d c52h6, by ACROBiosystems (2 mentions) Spd s52h6, by ACROBiosystems (2 mentions) Spd m121, by ACROBiosystems (2 mentions) Cr3022, by Creative Biolabs (2 mentions) Anti human igg fc hrp, by Southern Biotech (2 mentions) Hrp labeled goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions) Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions) Goat anti human igg fc hrp, by Southern Biotech (1 mentions) Nun c5227, by ACROBiosystems (1 mentions)

8 protocols using ni nta hissorb 96 well plates

1

Anti-trimer ELISA for HIV Env

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The anti-trimer ELISA was performed as previously described [18] (link), [40] (link). Briefly, supernatants containing His-tagged Envwt were diluted 1∶3 in TBS supplemented with 10% FCS and added for 2 h to pre-blocked Ni-NTA HisSorb 96-well plates (Qiagen, Venlo, the Netherlands). After three washes with TSM (20 mM Tris, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2), serially diluted animal sera treated and diluted as described above for the anti-gp120 ELISA, were then added for 2 h followed by three washes with TSM 0.05% Tween-20 (TSM-T). HRP-labeled goat anti-rabbit IgG or goat anti-mouse IgG (all from Jackson ImmunoResearch) was added for 1 h at a 1∶3000 dilution (final concentration 0.33 µg/ml) in TSM plus 5% BSA, followed by 5 washes with TSM-T. The subsequent steps of the assay were identical to those of the anti-gp120 ELISA.
Isik G., Sliepen K., van Montfort T, & Sanders R.W. (2014). Enhanced Immunogenicity of HIV-1 Envelope gp140 Proteins Fused to APRIL. PLoS ONE, 9(9), e107683.
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2

ELISA-Based Antibody Binding Assay

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Pure HEK293T cell supernatants containing BG505 SOSIP.664, or PGT145 affinity-purified BG505 SOSIP.664 and BG505 SOSIP.MPER (0.5 μg ml−1), all with a C-terminal 8 histidine-tag were immobilized on Ni-NTA HisSorb 96-well plates (Qiagen) (100 μl per well) for 2 h at RT. After three washing steps with Tris-buffered saline (TBS), antibodies were serially diluted in 2% skim milk and added for 2 h. Subsequently, HRP labelled goat anti-human Abs (Jackson ImmunoResearch) were added for 1 h in TBS, 2% skimmed milk, followed by five washes with TBS, 0.05% Tween-20. Colorimetric detection was performed using a solution containing 1% 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich), 0.01% H2O2, 100 mM sodium acetate and 100 mM citric acid. ELISA curves were fitted using Prism 5.01 (GraphPad).
Lee J.H., Leaman D.P., Kim A.S., Torrents de la Peña A., Sliepen K., Yasmeen A., Derking R., Ramos A., de Taeye S.W., Ozorowski G., Klein F., Burton D.R., Nussenzweig M.C., Poignard P., Moore J.P., Klasse P.J., Sanders R.W., Zwick M.B., Wilson I.A, & Ward A.B. (2015). Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike. Nature Communications, 6, 8167.
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3

Anti-gp120 Antibody Titration by ELISA

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Anti-gp120 Ab titers were measured by D7324-capture ELISA as described previously (35 (link),44 (link),47 (link)). Reactivity of Env proteins with MAbs or receptor mimics were measured by anti-trimer ELISA as described previously using Ni-NTA HisSorb 96-well plates (Qiagen, Venlo, The Netherlands)(10 (link),34 (link)–36 (link),47 (link)). Equal input levels of proteins were verified by SDS-PAGE followed by western blot analysis. All ELISA data are representative of at least three independent experiments, each using Env proteins derived from independent transfections. Endpoint titers were calculated using GraphPad Prism (version 5.03) by determining the serum dilution at which the optical density (OD450) was three times above the background OD450 signal (without animal sera).
Isik G., van Montfort T., Chung N.P., Moore J.P, & Sanders R.W. (2014). Autoantibodies induced by chimeric cytokine - HIV envelope glycoprotein immunogens. Journal of immunology (Baltimore, Md. : 1950), 192(10), 4628-4635.
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4

SARS-CoV-2 Antibody ELISA Quantification

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Ni-NTA HisSorb 96-well plates (Qiagen) were coated with 50 μl of either His-tagged RBD (Acro Biosystems, SPD-S52H6, AA 306-527) or His-tagged NTD (Acro Biosystems, S1D-C52H6, AA13-303) at a concentration of 2 μg/ml overnight at 4 °C; all subsequent steps were performed at room temperature. Plates were blocked with 3% milk; plasma was diluted 1:500 in PBS for RBD ELISA and 1:1500 for NTD ELISA prior to adding 100 μl per well for 2 h. Plates were washed with PBS-T three times, followed by incubation with secondary anti-human IgG Fc HRP (Southern Biotech) at 1:4000 for 1 h. Plates were washed three additional times with PBS-T, followed by addition of TMB substrate for 10 min. The reaction was stopped with the addition of 3 M HCl; and plates were read at an OD 450. Anti-RBD antibody CR3022 (Creative Biolabs) was used to make a standard curve with 1:2 dilutions in the range of 0.5 μg/ml to 0.03 μg/ml, utilizing a 4-parameter fit to interpolate sample concentrations. Anti-NTD antibody SPD-M121 (Acro Biosystems) was used to make a standard curve with 1:2 dilutions in the range of 0.5 μg/ml to 0.03 μg/ml, utilizing a 4-parameter fit to interpolate sample concentrations.
Ozer E.A., Simons L.M., Adewumi O.M., Fowotade A.A., Omoruyi E.C., Adeniji J.A., Olayinka O.A., Dean T.J., Zayas J., Bhimalli P.P., Ash M.K., Maiga A.I., Somboro A.M., Maiga M., Godzik A., Schneider J.R., Mamede J.I., Taiwo B.O., Hultquist J.F, & Lorenzo-Redondo R. (2022). Multiple expansions of globally uncommon SARS-CoV-2 lineages in Nigeria. Nature Communications, 13, 688.
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5

BG505 SOSIP.664 and BG505 SOSIP.MPER ELISA

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Pure HEK293T cell supernatants containing BG505 SOSIP.664, or PGT145 affinity purified BG505 SOSIP.664 and BG505 SOSIP.MPER (0.5 μg/mL), all with a C-terminal 8 histidine-tag were immobilized on Ni-NTA HisSorb 96-well plates (Qiagen) (100 μL/well) for 2 hours at room temperature. After three washing steps with Tris-Buffered Saline (TBS), antibodies were serially diluted in 2% skim milk and added for 2 hours. Subsequently, HRP labeled goat-anti-human Abs (Jackson Immunoresearch) were added for 1 hour in TBS, 2% skimmed milk, followed by five washes with TBS, 0.05% Tween-20. Colorimetric detection was performed using a solution containing 1% 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich), 0.01% H2O2, 100 mM sodium acetate and 100 mM citric acid. ELISA curves were fitted using Prism 5.01 (Graphpad).
Lee J.H., Leaman D.P., Kim A.S., Torrents de la Peña A., Sliepen K., Yasmeen A., Derking R., Ramos A., de Taeye S.W., Ozorowski G., Klein F., Burton D.R., Nussenzweig M.C., Poignard P., Moore J.P., Klasse P.J., Sanders R.W., Zwick M.B., Wilson I.A, & Ward A.B. (2015). Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike. Nature Communications, 6, 8167.
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6

SARS-CoV-2 Protein ELISA Immunoassay

Cited 3 times
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Ni-NTA HisSorb 96-well plates (Qiagen) were coated with 50 μL of either His-tagged RBD (Acro Biosystems, SPD-S52H6, AA 306–527), His-tagged NTD (Acro Biosystems, S1D-C52H6, AA13-303) or His-tagged Nucleocapsid (Acro Biosystems, NUN-C5227, AA1-419) at a concentration of 2 μg/mL overnight at 4°C according to previously published methods.31 (link)
,38 (link)
,39 (link) In brief all subsequent steps were performed at room temperature. Plates were blocked with 3% milk; plasma was diluted 1:500 in PBS for RBD ELISA, 1:1500 for NTD ELISA, and 1:500 for Nucleocapsid ELISA prior to adding 100 μL per well for 2 h. Plates were washed with PBS-T (PBS containing 0.1% Triton X-100) 3 times, followed by incubation with secondary goat anti-human IgG Fc HRP (Southern Biotech) at 1:4000 for 1 h. Plates were washed 3 additional times with PBS-T, followed by addition of SigmaFAST OPD substrate for 10 min. The reaction was stopped with the addition of 3M HCl; and plates were read at an OD 450.
Ash M.K., Bhimalli P.P., Cho B.K., Mattamana B.B., Gambut S., Tarhoni I., Fhied C.L., Reyes A.F., Welninski S.J., Arivalagan J., Negrão F., Goel R., Beck T.L., Hope T.J., Sha B.E., Goo Y.A., Al-Harthi L., Mamede J.I., Borgia J.A., Kelleher N.L, & Schneider J.R. (2022). Bulk IgG glycosylation predicts COVID-19 severity and vaccine antibody response. Cell Reports, 41(11), 111799.
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7

Ni-NTA ELISA for Analyzing Antibody Binding to BG505 SOSIP

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The procedure for the Ni-NTA ELISA has been previously described (Bontjer et al., 2010 (link)). Briefly, 0.1 µg/ml of His-tagged BG505 SOSIP gp140 was added in PBS to Ni-NTA His Sorb 96-well plates (Qiagen) for 1 hr at 37°C. Wells were washed 4 times with PBS 0.05% Tween 20. MAbs were added at a serial dilution in 1% BSA in PBS for 2 hrs at 37°C. Wells were washed and anti-human IgG F(ab’)2 -HRP was added at 1:5,000 dilution for 45 min at room temperature. Wells were washed and 1:1 mixed TMB substrate solution (Pierce) was added for colorimetric detection. The colorimetric reaction was stopped using 2N H2SO4 and absorption was measured at 450 nm. Uncleaved tagged BG505 SOSIP was generated by replacing the optimized cleavage site (RRRRRR) by an uncleavable motif (SEKS) (Ringe et al., 2013 (link)). The ELISA for uncleaved D7324-tagged BG505 SOSIP trimers and corresponding cleaved D7324-tagged BG505 SOSIP was previously described (Ringe et al., 2013 (link)). In Figure 2C, MAb binding to uncleaved D7324-tagged BG505 SOSIP or cleaved His-tagged BG505 SOSIP is shown. The data for cleaved D7324-tagged BG505 SOSIP is not shown but is comparable to Histagged BG505 SOSIP.
Falkowska E., Le K.M., Ramos A., Doores K.J., Lee J.H., Blattner C., Ramirez A., Derking R., van Gils M.J., Liang C.H., Mcbride R., von Bredow B., Shivatare S.S., Wu C.Y., Chan-Hui P.Y., Liu Y., Feizi T., Zwick M.B., Koff W.C., Seaman M.S., Swiderek K., Moore J.P., Evans D., Paulson J.C., Wong C.H., Ward A.B., Wilson I.A., Sanders R.W., Poignard P, & Burton D.R. (2014). Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the pre-fusion conformation of the gp41 protein on cleaved Envelope trimers. Immunity, 40(5), 657-668.
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8

SARS-CoV-2 RBD and NTD Antibody ELISA

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Ni-NTA HisSorb 96-well plates (Qiagen) were coated with 50ul of either His-tagged RBD or His-tagged NTD (Acro Biosystems) at a concentration of 2ug/ml overnight at 4°C; all subsequent steps were performed at room temperature. Plates were blocked with 3% milk; plasma was diluted 1:500 in PBS for RBD ELISA and 1:1500 for NTD ELISA prior to adding 100ul per well for 2 hours. Plates were washed with PBS-T 3 times, followed by incubation with secondary anti-human IgG Fc HRP (Southern Biotech) at 1:4000 for 1hr. Plates were washed 3 additional times with PBS-T, followed by addition of TMB substrate for 10min. The reaction was stopped with the addition of 3M HCl; and plates were read at an OD 450. Anti-RBD antibody CR3022 (Creative Biolabs) was used to make a standard curve with 1:2 dilutions in the range of 0.5ug/ml to 0.03ug/ml, utilizing a 4-parameter fit to interpolate sample concentrations. Anti-NTD antibody SPD-M121 (Acro Biosystems) was used to make a standard curve with 1:2 dilutions in the range of 0.5ug/ml to 0.03ug/ml, utilizing a 4-parameter fit to interpolate sample concentrations.
Ozer E.A., Simons L.M., Adewumi O.M., Fowotade A.A., Omoruyi E.C., Adeniji J.A., Dean T.J., Zayas J., Bhimalli P.P., Ash M.K., Godzik A., Schneider J.R., Mamede J.I., Taiwo B.O., Hultquist J.F, & Lorenzo-Redondo R. (2021). Coincident rapid expansion of two SARS-CoV-2 lineages with enhanced infectivity in Nigeria. medRxiv.
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