Log In Sign up
The largest database of trusted experimental protocols

Alkaline phosphatase conjugated streptavidin

Manufactured by Thermo Fisher Scientific
Visit Supplier
Sourced in United States

Alkaline phosphatase-conjugated streptavidin is a protein complex composed of streptavidin, a protein derived from the bacterium Streptomyces avidinii, and alkaline phosphatase, an enzyme that catalyzes the hydrolysis of phosphate esters. The complex is used as a detection reagent in various bioanalytical techniques, such as enzyme-linked immunosorbent assays (ELISA) and Western blotting, where it can bind to biotinylated molecules and provide a colorimetric or chemiluminescent signal.

Automatically generated - may contain errors

Lab products found in correlation

Pnpp substrate, by Thermo Fisher Scientific (1 mentions) Nunc maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions) Hematoxylin, by Bio-Optica (1 mentions) Ab828, by Abcam (1 mentions) Dmrd optical microscope, by Leica (1 mentions) Ab16074, by Abcam (1 mentions) Vulcan fast red chromogen, by Biocare Medical (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) P nitrophenyl phosphate, by Merck Group (1 mentions) Imark microplate reader, by Bio-Rad (1 mentions)

6 protocols using alkaline phosphatase conjugated streptavidin

1

Quantifying Influenza Virus Infection in Mice

Check if the same lab product or an alternative is used in the 5 most similar protocols
Influenza A/PR8/34 (PR8) was used at 15,000 viral foci units (VFU) for intranasal infection and at 1 × 109 VFU for intraperitoneal infection. In Figure 7a–d, the i.p. infections were repeated for 6 days at 1 × 108 VFU, 1 × 108 VFU, 1 × 109 VFU, 1 × 109 VFU, 1 × 108 VFU and 1 × 108 VFU. Influenza X31 was used at 1.25 × 104 VFU for primary infection and 1.25 × 105 VFU for secondary infections. Mice were anesthetized with vaporized isofluorane and infected intranasally with 100 μl of inoculum or intratracheally with 75 μl inoculum. Viral foci assays were performed by homogenizing lung samples in zero–serum medium with 4 μg/ml trypsin and performing 5–fold serial dilutions in the same media. 100 μl of each sample was used to infect MDCK cell monolayers. After 16 h of culture, MDCK cells were fixed with 80% acetone and infected cells were detected using an antibody against NP (Clone A3. Millipore–Sigma). Spots were revealed using alkaline phosphatase–conjugated streptavidin (Life Technologies) and BCIP/NBT substrate (Moss Substrates Inc.). Samples were plated in duplicates and the mean foci count was used to calculate the VFU per ml of sample.
Allie S.R., Bradley J.E., Mudunuru U., Schultz M.D., Graf B.A., Lund F.E, & Randall T.D. (2018). The establishment of resident memory B cells in the lung requires local antigen encounter. Nature immunology, 20(1), 97-108.
+ Open protocol
+ Expand
2

Quantifying Influenza Virus Infection in Mice

Check if the same lab product or an alternative is used in the 5 most similar protocols
Influenza A/PR8/34 (PR8) was used at 15,000 viral foci units (VFU) for intranasal infection and at 1 × 109 VFU for intraperitoneal infection. In Figure 7a–d, the i.p. infections were repeated for 6 days at 1 × 108 VFU, 1 × 108 VFU, 1 × 109 VFU, 1 × 109 VFU, 1 × 108 VFU and 1 × 108 VFU. Influenza X31 was used at 1.25 × 104 VFU for primary infection and 1.25 × 105 VFU for secondary infections. Mice were anesthetized with vaporized isofluorane and infected intranasally with 100 μl of inoculum or intratracheally with 75 μl inoculum. Viral foci assays were performed by homogenizing lung samples in zero–serum medium with 4 μg/ml trypsin and performing 5–fold serial dilutions in the same media. 100 μl of each sample was used to infect MDCK cell monolayers. After 16 h of culture, MDCK cells were fixed with 80% acetone and infected cells were detected using an antibody against NP (Clone A3. Millipore–Sigma). Spots were revealed using alkaline phosphatase–conjugated streptavidin (Life Technologies) and BCIP/NBT substrate (Moss Substrates Inc.). Samples were plated in duplicates and the mean foci count was used to calculate the VFU per ml of sample.
Allie S.R., Bradley J.E., Mudunuru U., Schultz M.D., Graf B.A., Lund F.E, & Randall T.D. (2018). The establishment of resident memory B cells in the lung requires local antigen encounter. Nature immunology, 20(1), 97-108.
+ Open protocol
+ Expand
3

Measurement of TDI-specific IgG and IgE

Check if the same lab product or an alternative is used in the 5 most similar protocols
TDI-specific IgG and IgE levels in serum samples from each worker at baseline, 6-, and 12-months post-employment were measured using previously described enzyme-linked immunosorbent assays (ELISA) [Ye, et al. 2006 (link)]. Briefly, TDI-albumin conjugates (10 μg/ml) prepared by mixed phase (vapor/liquid) exposure methods were used to coat 96-well NUNC Maxisorp ELISA plates (Thermo Fisher Scientific Inc., Waltham, MA), followed by blocking with 3% dry milk in phosphate buffered saline (PBS). Workers’ sera were diluted in 3% milk + PBS + 0.05% Tween 20 and TDI-specific IgG was detected with horseradish peroxidase linked anti-human IgG antibodies from Pharmingen (San Jose, CA), and expressed as an end-titer. For detecting TDI-specific IgE, plates were developed with biotinylated goat anti-human IgE (Bethyl; Montgomery, TX) followed by alkaline phosphatase conjugated streptavidin, and pNPP substrate from Thermo Fisher Scientific Inc.
Gui W., Wisnewski A.V., Neamtiu I., Gurzau E., Sparer J.A., Stowe M.H., Liu J., Slade M.D., Rusu O.A, & Redlich C.A. (2014). Inception Cohort Study of Workers Exposed to Toluene Diisocyanate at a Polyurethane Foam Factory: Initial One-Year Follow-up. American journal of industrial medicine, 57(11), 1207-1215.
+ Open protocol
+ Expand
4

Immunohistochemical Analysis of Mammary Tumors

Check if the same lab product or an alternative is used in the 5 most similar protocols
Whole mounts were prepared as previously described [15 (link)]. Mammary tumors were fixed in formalin and embedded in paraffin. hematoxylin and eosin staining (H&E) was used to assess histology. Infiltration by CD3+ and perforin-positive cells was determined through immunohistochemistry with anti-CD3 (Ab828; Abcam, Cambridge, UK) and anti-perforin (Ab16074, Abcam) antibodies as previously described [16 (link)]. Immunostaining was developed using alkaline phosphatase-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA) and vulcan fast red chromogen (Biocare Medical, Concord, CA, USA). Slides were counterstained with hematoxylin (BioOptica, Milan, Italy) and images were acquired by Leica DMRD optical microscope (Leica, Milton Keynes, UK).
Croci S., Nanni P., Palladini A., Nicoletti G., Grosso V., Benegiamo G., Landuzzi L., Lamolinara A., Ianzano M.L., Ranieri D., Dall’Ora M., Iezzi M., De Giovanni C, & Lollini P.L. (2015). Interleukin-15 is required for immunosurveillance and immunoprevention of HER2/neu-driven mammary carcinogenesis. Breast Cancer Research : BCR, 17(1), 70.
+ Open protocol
+ Expand
5

SARS-CoV-2 and HIV-1 Spike Protein ELISpot Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
For ELISpot assays, MSIP 96-well plates were from Merck Millipore (multiscreen filter plates; Burlington, MA, USA), and anti-mouse or anti-monkey IFN-γ or IL-4 capture and detection antibodies were all from U-CyTech (Utrecht, Netherlands). Lymphocytes (from mouse spleens/lungs or macaque PBMC) were plated in duplicate at appropriate cell densities and cultured for 18−20 h at 37°C, 5% CO2 with 15-mer peptide pools covering the entire SARS-CoV-2 Spike protein referred to as S-1 or S-2 (JPT PepMix; JPT, Berlin, Germany) or covering HIV-1 gag protein (Elabscience, distributed by Tema Ricerca Srl, Italy). Stimulation conditions were 1 (for mouse lymphocytes) or 3 μg/mL (for monkey’s PBMCs) final single peptide concentration or the equivalent amount of DMSO (Sigma), the peptide diluent, as negative control. Cytokine production and spot formation were detected with alkaline phosphatase-conjugated streptavidin and NBT/BCIP alkaline phosphatase substrate (Thermo Scientific). The number of spots per well, directly related to the precursor frequency of antigen-specific T cells, was counted and analyzed with a CTL ImmunoSpot reader (ImmunoSpot, Shaker Heights, OH, USA). Data are expressed as cytokine SFCs per million splenocytes, lung lymphocytes, or PBMC and are shown upon subtraction of DMSO background.
Capone S., Raggioli A., Gentile M., Battella S., Lahm A., Sommella A., Contino A.M., Urbanowicz R.A., Scala R., Barra F., Leuzzi A., Lilli E., Miselli G., Noto A., Ferraiuolo M., Talotta F., Tsoleridis T., Castilletti C., Matusali G., Colavita F., Lapa D., Meschi S., Capobianchi M., Soriani M., Folgori A., Ball J.K., Colloca S, & Vitelli A. (2021). Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19. Molecular Therapy, 29(8), 2412-2423.
+ Open protocol
+ Expand
6

Quantifying Antibody Responses to Hemagglutinin

Check if the same lab product or an alternative is used in the 5 most similar protocols
Hemagglutinin (HA)-specific IgG and IgA antibodies in serum or nasal wash samples were quantified by enzyme-linked immunosorbent assay (ELISA). Recombinant trimeric HA, produced by the method of Stevens et al.,49 (link) was used as the coating antigen. HA-specific IgA antibodies were detected using biotin-conjugated goat anti-mouse IgA antibodies (Kirkegaard & Perry Laboratories, USA) and alkaline phosphatase-conjugated streptavidin (Thermo Fisher Scientific, USA). The detection reaction was initiated by the addition of p-nitrophenylphosphate (Sigma-Aldrich, USA) in 10 mM diethanolamine (pH: 9.8) containing 0.5 mM MgCl2. Absorbance at 405 nm was measured using an iMark Microplate Reader (BIO-RAD, Hercules, USA). Pooled nasal wash samples collected from hyperimmune mice was used as a standard for the quantification of nasal IgA. ELISA was performed using serial two-fold dilutions of standard serum and nasal wash (NW) (from 1 : 100 and 1 : 1, respectively) as described above. The reciprocal of highest dilution rate of sample showing positive reaction was defined as ELISA unit. The threshold value for positive reaction was defined as mean ± 3SD of serially diluted serum or NW samples collected from a non-vaccinated BALB/c mouse.
Tazaki T., Tabata K., Ainai A., Ohara Y., Kobayashi S., Ninomiya T., Orba Y., Mitomo H., Nakano T., Hasegawa H., Ijiro K., Sawa H., Suzuki T, & Niikura K. (2018). Shape-dependent adjuvanticity of nanoparticle-conjugated RNA adjuvants for intranasal inactivated influenza vaccines. RSC Advances, 8(30), 16527-16536.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!