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Np20 bsa

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The NP20-BSA is a type of laboratory equipment used for surface analysis. It is a chip that contains a gold surface coated with bovine serum albumin (BSA) for use in surface plasmon resonance (SPR) experiments. The NP20-BSA chip is designed to provide a stable and consistent surface for the study of biomolecular interactions.

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Lab products found in correlation

Pnpp substrate, by Merck Group (3 mentions) Np4 bsa, by Biosearch Technologies (3 mentions) Microplate reader, by Molecular Devices (3 mentions) Anti isotype specific antibodies, by BD (2 mentions) Streptavidin ap, by Southern Biotech (2 mentions) Np2 bsa, by Biosearch Technologies (2 mentions) Tmb substrate, by BioLegend (2 mentions) Np7 bsa, by Biosearch Technologies (2 mentions) Sandwich elisa, by BD (1 mentions) Multi screen ha filtration, by Merck Group (1 mentions) Ba 2020, by Vector Laboratories (1 mentions) Avidin d hrp, by Vector Laboratories (1 mentions) Elispot aec substrate, by BD (1 mentions) Streptavidin hrp, by Southern Biotech (1 mentions) Aec staining kit, by Merck Group (1 mentions) Sigmafast opd tablet, by Merck Group (1 mentions) Immunospot s6 analyzer, by Cellular Technology (1 mentions) Multiscreen ha mixed cellulose ester plates, by Merck Group (1 mentions) Hrp conjugated anti mouse igm or igg3 antibodies, by Southern Biotech (1 mentions) Iscove modified dulbecco media (imdm), by Merck Group (1 mentions)

16 protocols using np20 bsa

1

Sandwich ELISA for Antibody Analysis

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Sandwich ELISA for measuring preimmune sera antibodies using anti-isotype–specific antibodies (BD Pharmingen) to capture IgM, IgG1, IgG2b, or IgG3 were done as described (Zahn et al., 2013 (link)). Antigen-specific antibodies were captured from immunized mice sera by coating ELISA plates with NP20-BSA (Biosearch Technologies) or CGG (100 ng/well; Biosearch Technologies) followed by the detection of IgG1, as described (Zahn et al., 2013 (link)). Sodium thiocyanate NaSCN displacement ELISA to measure antibody affinity/avidity was performed as described (Zahn et al., 2013 (link)), on plates coated as above. Sera were previously titrated by antigen-specific ELISA to choose a working dilution that ensured similar levels of antigen-specific antibodies across samples. Relative affinity values were calculated as described (Zahn et al., 2013 (link)).
Litzler L.C., Zahn A., Dionne K.L., Sprumont A., Ferreira S.R., Slattery M.R., Methot S.P., Patenaude A.M., Hébert S., Kabir N., Subramani P.G., Jung S., Richard S., Kleinman C.L, & Di Noia J.M. (2023). Protein arginine methyltransferase 1 regulates B cell fate after positive selection in the germinal center in mice. The Journal of Experimental Medicine, 220(9), e20220381.
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2

NP-Specific ELISA and ELISpot Assays

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Assays were done in Millipore Multi-Screen HA filtration 96-well plates (no. MSIPN4W). IgM, IgG1, and IgG3 coating Abs were the same as for ELISA. Secondary Abs were purchased from Vector Laboratories for detecting IgM (no. BA-2020) and IgG (no. BA-9200), followed by HRP-Avidin D (no. A-2004; Vector Laboratories). For NP-specific assays, plates were coated with NP(>20)-BSA (no. N-5050H-10; Biosearch Technologies). PBS plus 2% BSA was used as blocking buffer, ELISpot AEC substrate (no. 551951; BD Biosciences) was used to develop. Plates were scanned on a CTL ImmunoSpot S6 Entry instrument and analyzed using CTL ImmunoSpot software, version 7.0.9.5. All samples were done over a 12-step dilution series. Plotted spots per 106 cells were determined by averaging spots per 106 cells over a minimum of three different dilutions, with attention paid to using wells within the linear range. Counting parameters were held constant across all plates of the same assay type. Quality control of automated counting was performed on every plate by visual inspection.
Pucella J.N., Cols M., Yen W.F., Xu S, & Chaudhuri J. (2019). The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses. Journal of immunology (Baltimore, Md. : 1950), 202(5), 1383-1396.
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3

NP-Specific Antibody Responses in μMT Mice

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For ELISA, serum was prepared from the blood of μMT chimeric mice. ELISA plates (Nunc Maxisorp) were coated with NP20-BSA (Biosearch Technologies) and blocked with 1% BSA in PBS. Serial dilutions of serum samples (0.1% BSA/PBS) were added and incubated overnight. NP-specific IgM and IgG3 antibodies were detected using biotinylated anti-mouse IgM- or IgG3-specific immunoglobulins followed by streptavidin-HRP (Southern Biotech) and developed with SIGMAFAST OPD tablets (Sigma-Aldrich). Absorbance values at 490 nm were determined and used to calculate endpoint titers.
For ELISpot, serial dilution of splenocyte suspensions of μMT chimeric mice were added to NP20-BSA-coated MultiScreen HA mixed cellulose ester plates (Millipore, Watford, UK) previously washed and blocked with IMDM (Sigma-Aldrich) freshly supplemented with 10% FCS, 2 mM GlutaMAX and 50 μM 2-mercaptoethanol. Upon overnight incubation, cells secreting anti-NP antibody (ASC) were visualized with HRP-conjugated anti-mouse IgM or IgG3 antibodies (Southern Biotech) followed by AEC staining Kit (Sigma-Aldrich). The numbers of ASCs were quantified using Immunospot S6 Analyzer (Cellular Technology Limited).
Salerno F., Howden A.J., Matheson L.S., Gizlenci Ö., Screen M., Lingel H., Brunner-Weinzierl M.C, & Turner M. (2023). An integrated proteome and transcriptome of B cell maturation defines poised activation states of transitional and mature B cells. Nature Communications, 14, 5116.
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4

ELISA for Total and Antigen-Specific Ig

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ELISA for total and antigen-specific immunoglobulin was performed as previously described (18 (link)). Briefly, half area plates (Nunc) were coated with αIg antibodies, 5 μg/mL NP4-BSA or NP20-BSA (Biosearch Technologies) in PBS overnight at 4˚C, washed 4X with PBS + 0.05% Tween20, and blocked with PBS + 1% BSA for 2 hours at room temperature. Serial dilutions of serum in PBS + 1% BSA from NP-OVA immunized mice were applied for 3h at room temperature. Plates were washed 4X with PBS + 0.05% Tween20 and incubated with 1:500 alkaline phosphatase-conjugated Ig isotype-specific antibodies (Southern Biotech). Bound antibody was detected with pNPP substrate (Sigma-Aldrich), and absorbance was measured at 405 nm with a microplate reader (Molecular Devices). For total Ig ELISAs, wells were coated with 5 μg/mL anti-mouse Ig-isotype-specific antibodies (Southern Biotech) instead of NP-BSA. Values were calculated according to Ig standards (Southern Biotech) or reference serum from NP-OVA immunized mice.
Johansen K.H., Golec D.P., Huang B., Park C., Thomsen J.H., Preite S., Cannons J.L., Garçon F., Schrom E.C., Courreges C., Veres T.Z., Harrison J., Nus M., Phelan J.D., Bergmeier W., Kehrl J., Okkenhaug K, & Schwartzberg P.L. (2022). A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1–mediated adhesion in T cells. Science signaling, 15(743), eabl9169.
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5

ELISPOT Assay for IgG1-Secreting Cells

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One million splenocytes were seeded in each well of the MultiScreen filter plate (Milipore, Darmstadt, Germany) coated with NP20-BSA (Biosearch Technologies, Novato, CA, USA) and blocked with BSA for incubation. After washing, the plate was incubated with biotin-conjugated anti-IgG1 antibodies (1/1000, SouthernBiotech, Birmingham, AL, USA) for 1 h at room temperature, and with streptavidin-AP (1/1000, SouthernBiotech, Birmingham, AL, USA) for another 0.5 h. Then, the antibody-secreting cells were visualized using substrates (Mabtech, Cincinnati, OH, USA).
Wang W., Huang H., Jiang H., Tian C., Tang Y., Gan D., Wen X., Song Z., He Y., Ou X, & Fang L. (2022). A Cross-Tissue Investigation of Molecular Targets and Physiological Functions of Nsun6 Using Knockout Mice. International Journal of Molecular Sciences, 23(12), 6584.
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6

Serum Antibody and BAFF Quantification

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To measure antibody or BAFF levels in the serum, blood was withdrawn from the tail vein. NP+ IgM and IgG1 serum titers were measured by ELISA using Maxisorp plates (Nunc) coated with 5 µg/ml NP20-BSA (Biosearch Technologies) as previously described (Hartweger et al., 2014 (link)). BAFF serum titers were detected using the Mouse BAFF ELISA Kit (Tnfsf13b; Abcam).
Müller-Winkler J., Mitter R., Rappe J.C., Vanes L., Schweighoffer E., Mohammadi H., Wack A, & Tybulewicz V.L. (2020). Critical requirement for BCR, BAFF, and BAFFR in memory B cell survival. The Journal of Experimental Medicine, 218(2), e20191393.
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7

Sandwich ELISA for Antibody Measurement

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Sandwich ELISA for measuring pre-immune sera antibodies were done using plates coated with anti-isotype-specific antibodies (BD Pharmingen) to capture IgM, IgG1, IgG2b, or IgG3. Antigen-specific antibodies were captured from immunized mice sera in ELISA plates coated with NP20-BSA (Biosearch Technologies) followed by the detection of IgG1. Detection was as described62 (link). Briefly, antibodies were detected with the corresponding biotinylated anti-Ig (BD Pharmigen) followed by HRP-conjugated streptavidin (1:5000; Thermo Scientific) and developed using 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (Sigma).
Litzler L.C., Zahn A., Meli A.P., Hébert S., Patenaude A.M., Methot S.P., Sprumont A., Bois T., Kitamura D., Costantino S., King I.L., Kleinman C.L., Richard S, & Di Noia J.M. (2019). PRMT5 is essential for B cell development and germinal center dynamics. Nature Communications, 10, 22.
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8

NP-BSA ELISPOT Assay for Antibody-Secreting Cells

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ELISPOT assay was performed as described previously55 (link). In brief, MultiScreen filter plates with 0.45 μm PVDF membrane (Milipore) were coated with 1 μg per well NP20-BSA (Biosearch Technologies) in PBS overnight at 4°C and blocked with 2% BSA in PBS for 2 h at room temperature. One million splenocytes or three million BM cells were seeded in each well, and then the plate was incubated at 37°C for 2 h. After washing, the plate was incubated with Biotinylated anti-IgM (1: 1000, SouthernBiotech, 1140-08) or anti-IgG1 (1: 1000, SouthernBiotech, 1144-08) antibodies for 1 h at room temperature, followed by streptavidin-AP (1:1000, SouthernBiotech, 7105-04) incubation for 0.5 h at room temperature. The substrates (MABTECH) were added to each well for visualization of antibody-secreting cells.
Huang H., Li Y., Zhang G., Ruan G.X., Zhu Z., Chen W., Zou J., Zhang R., Wang J., Ouyang Y., Xu S, & Ou X. (2023). The RNA-binding protein hnRNP F is required for the germinal center B cell response. Nature Communications, 14, 1731.
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9

ELISA for Antigen-Specific Immunoglobulin

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ELISA for total and antigen-specific immunoglobulin was performed as previously described22 (link). Plates (Costar) were coated ON with 5 μg/ml anti-Ig antibodies or NP20-BSA and NP4-BSA (Biosearch Technologies) in PBS at 4°C. Plates were blocked with 1% BSA/PBS, serial dilutions of sera were applied and incubated at 20°C (room temperature) for 2–3 h. Subsequently, plates were washed (with PBS/0.05% Tween20) and incubated with alkaline phosphatase (AP)-conjugated anti-mouse Ig isotype-specific antibodies followed by pNPP substrate (Sigma-Aldrich) as detection reagents. Absorbance was measured at 405nm with a microplate reader (Molecular Devices) and values were calculated according to Ig standards or to reference serum from wild-type mice immunized twice with NP19-OVA (expressed in arbitrary units, AU). Ratios between NP4- versus NP20-binding antibodies were calculated as an estimate for affinity maturation. Fecal IgA ELISA were performed as previously described50 (link): fresh stools were collected, dissolved in PBS (100 μl/0.01 g), centrifuged, and IgA concentrations determined by ELISA on the recovered supernatants. Antibodies and immunoglobulin standards were purchased through Southern Biotech.
Preite S., Cannons J.L., Radtke A.J., Vujkovic-Cvijin I., Gomez-Rodriguez J., Volpi S., Huang B., Cheng J., Collins N., Reilley J., Handon R., Dobbs K., Huq L., Raman I., Zhu C., Li Q.Z., Li M.O., Pittaluga S., Uzel G., Notarangelo L.D., Belkaid Y., Germain R.N, & Schwartzberg P.L. (2018). Hyperactivated PI3Kδ promotes self and commensal reactivity at the expense of optimal humoral immunity. Nature immunology, 19(9), 986-1000.
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10

ELISA for Antigen-Specific Immunoglobulin

Cited 1 time
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ELISA for total and antigen-specific immunoglobulin was performed as previously described22 (link). Plates (Costar) were coated ON with 5 μg/ml anti-Ig antibodies or NP20-BSA and NP4-BSA (Biosearch Technologies) in PBS at 4°C. Plates were blocked with 1% BSA/PBS, serial dilutions of sera were applied and incubated at 20°C (room temperature) for 2–3 h. Subsequently, plates were washed (with PBS/0.05% Tween20) and incubated with alkaline phosphatase (AP)-conjugated anti-mouse Ig isotype-specific antibodies followed by pNPP substrate (Sigma-Aldrich) as detection reagents. Absorbance was measured at 405nm with a microplate reader (Molecular Devices) and values were calculated according to Ig standards or to reference serum from wild-type mice immunized twice with NP19-OVA (expressed in arbitrary units, AU). Ratios between NP4- versus NP20-binding antibodies were calculated as an estimate for affinity maturation. Fecal IgA ELISA were performed as previously described50 (link): fresh stools were collected, dissolved in PBS (100 μl/0.01 g), centrifuged, and IgA concentrations determined by ELISA on the recovered supernatants. Antibodies and immunoglobulin standards were purchased through Southern Biotech.
Preite S., Cannons J.L., Radtke A.J., Vujkovic-Cvijin I., Gomez-Rodriguez J., Volpi S., Huang B., Cheng J., Collins N., Reilley J., Handon R., Dobbs K., Huq L., Raman I., Zhu C., Li Q.Z., Li M.O., Pittaluga S., Uzel G., Notarangelo L.D., Belkaid Y., Germain R.N, & Schwartzberg P.L. (2018). Hyperactivated PI3Kδ promotes self and commensal reactivity at the expense of optimal humoral immunity. Nature immunology, 19(9), 986-1000.
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