Affinipure f ab 2 fragment goat anti mouse igg h l

Manufactured by Jackson ImmunoResearch

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AffiniPure F (ab′)2 Fragment Goat Anti-Mouse IgG (H + L) is a laboratory reagent that binds to the Fab region of mouse immunoglobulin G (IgG) antibodies. It is generated by enzymatic cleavage of the parent antibody, resulting in a divalent fragment that retains antigen-binding capabilities.

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AffiniPure goat anti-mouse IgG (H+L) (38 citations) Goat anti-mouse IgG (H+L) (36 citations) AffiniPure goat anti-mouse IgG (28 citations) AffiniPure F(ab’)2 fragment goat anti mouse IgG (7 citations) Cy™3 AffiniPure F(ab’)2 fragment goat anti-mouse IgG (H+L) (3 citations) Alexa Fluor 594-conjuated Affinipure F (ab’)2 fragment goat anti-mouse IgG (H+L) secondary antibody (2 citations) Peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti mouse IgG (H+L) (2 citations)

7 protocols using affinipure f ab 2 fragment goat anti mouse igg h l

Generation and Characterization of Zika and Usutu Virus Strains

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Two strains of infectious Zika viruses (1947 Uganda MR766 and 2013 French Polynesia HPF2013) were generated by transfection of 293T cells with two plasmids (pCDNA6.2 Zika MR766 Intron3115 HDVr MEG 070916 5 and pCDNA6.2 Zika HPF2013 3864,9388Intron HDVr MEG091316 2) as previously described [49 (link)]. The viruses were then propagated in Vero cells and titrated by plaque assay. All the antiviral assays were performed with ZIKV HPF2013 strain, unless otherwise stated.
Usutu virus (Strain: 3345 Isolate: Arb276) was isolated and produced by APHA (Animal & Plant Health Agency–GOV. UK) and kindly provided by the European Viral Archive Global (EVAg). It was propagated in Vero cells and titrated by means of the indirect immunoperoxidase staining procedure, by using a mouse monoclonal antibody direct to flavivirus protein E (D1-4G2-4-15 (4G2), Novus Biological) and a secondary antibody peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratories Inc., 872 W. Baltimore Pike, West Grove, PA 19390).

Francese R., Civra A., Donalisio M., Volpi N., Capitani F., Sottemano S., Tonetto P., Coscia A., Maiocco G., Moro G.E., Bertino E, & Lembo D. (2020). Anti-Zika virus and anti-Usutu virus activity of human milk and its components. PLoS Neglected Tropical Diseases, 14(10), e0008713.

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Serum Antibody Detection in Tumor-Challenged Mice

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At days 28-34th after tumor challenge, blood samples were obtained by sub-mandibular bleeding and serum stored at −80 °C until used.
To determine the presence of serum Abs by FACS, 2 × 10⁵ B16-F1 cells were incubated with 10% normal goat serum, centrifuged 5 min at 300 g at 4 °C, incubated for 60 min at 4 °C with serum dilutions, washed with PBS, incubated 30 min with a 1/100 dilution of RPE-goat anti-mouse Immunoglobulins (R0480, Dako), washed with PBS and fixed with 2% PFA. Mean Fluorescence Intensities (MFI) were analyzed in a FACScalibur cytometer.
For ELISA, B16-F1 cells and C57Bl/6 splenocyte monolayers on 96-well cell culture plates were fixed with 4% PFA for 15 min at room temperature, blocked with 100 μl 1% BSA in PBS for 1 h, incubated overnight at 4 °C with 75 μl of 1/200 serum dilutions in 0.1% BSA in PBS, washed 3 times with 150 μl 0.1% BSA in PBS, incubated with 100 μl 1/10000 dilution in 0.1% BSA in PBS of HRP-AffiniPure F (ab′)2 Fragment Goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch) and washed 3 times with 150 μl 0.1% BSA in PBS. TMB (Sigma) was used to detect HRP activity, and the reaction was stopped with 2 M H2SO4. Absorbance at 450 nm was assessed.

Keon S.M., Bentivegna S., Levy E.M., Marks M.S., Mantegazza A.R., Wainstok R, & Mordoh J. (2019). Syngeneic B16-F1 cells are more efficient than allogeneic Cloudman cells as antigen source in DC-based vaccination in the B16-F1 murine melanoma model. Vaccine, 37(35), 4947-4955.

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Anti-RSV and Anti-SARS-CoV-2 Antibody Protocol

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The oxysterol 27OHC complexed with 2-hydroxypropyl-β-cyclodextrin (2HP-βCD:27OHC) was kindly provided by Panoxyvir Ltd (Turin, Italy). The anti-RSV monoclonal antibody Ab35958 was purchased from Abcam (Cambridge, United Kingdom). The secondary antibody peroxidase-conjugated AffiniPure F (ab’)2 Fragment Goat Anti-Mouse IgG (H+L) was purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). The recombinant antibody mSIP-3022 against SARS-CoV-2 Spike was previously described [17 ].

Marcello A., Civra A., Milan Bonotto R., Nascimento Alves L., Rajasekharan S., Giacobone C., Caccia C., Cavalli R., Adami M., Brambilla P., Lembo D., Poli G, & Leoni V. (2020). The cholesterol metabolite 27-hydroxycholesterol inhibits SARS-CoV-2 and is markedly decreased in COVID-19 patients. Redox Biology, 36, 101682.

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Serum Antibody Detection in Tumor-Challenged Mice

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SARS-CoV-2 Antibody Detection Protocol

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Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (Saint Louis, MO). The mouse anti-coronavirus monoclonal antibody MAB9013 was purchased from Merck (Darmstadt, Germany). The secondary antibody peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG (H + L) was purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Milani M., Donalisio M., Bonotto R.M., Schneider E., Arduino I., Boni F., Lembo D., Marcello A, & Mastrangelo E. (2021). Combined in silico and in vitro approaches identified the antipsychotic drug lurasidone and the antiviral drug elbasvir as SARS-CoV2 and HCoV-OC43 inhibitors. Antiviral Research, 189, 105055.

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Antiviral Activity of Colostrum Compounds

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Antiviral activity of NHBC, HBC, or IgG against HRoV Wa, HRV408, HRV248, and NCDV was determined by focus reduction assay or plaque reduction assay. Assays of inhibition of rotavirus infectivity were carried out with confluent MA104 cell monolayers plated in 96-well trays, as described elsewhere (Civra et al., 2014 (link)). Cells were treated for 2 h at 37°C with serial dilutions of colostrum, at protein concentrations ranging from 0.02 to 3,340 µg of protein/mL in serum-free medium before virus addition. Infection with HRoV was performed at a multiplicity of infection (MOI) of 0.02 FFU/mL for 1 h at 37°C, in presence of the colostrums. Infected cells were washed with serum-free medium, fresh methanol extract was added, and cells were incubated in this medium at 37°C in a humidified incubator in 5% (vol/vol) CO₂ in 95% (vol/vol) air. After 16 h of incubation, infected cells were fixed with cold acetone-methanol (50:50), and viral titers determined by indirect immunostaining by using a mouse monoclonal antibody directed to human rotavirus VP6 (0036; Covalab, Villeurbanne, France), and the secondary antibody peroxidase-conjugated AffiniPure F(ab′)₂ Fragment Goat Anti-Mouse IgG (H⁺L) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA).

Civra A., Altomare A., Francese R., Donalisio M., Aldini G, & Lembo D. (2019). Colostrum from cows immunized with a veterinary vaccine against bovine rotavirus displays enhanced in vitro anti-human rotavirus activity. Journal of Dairy Science, 102(6), 4857-4869.

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Immunoblotting of HSV-2 Antigens

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Methylcellulose, crystal violet, dimethyl sulfoxide (DMSO), sodium dodecyl-sulphate (SDS), NP-40, sodium deoxycholate, a cocktail of protease inhibitors, Tween 20, glycine, and Triton X-100 were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The anti-HSV-2 ICP4 polyclonal antibody (ab96431) was from Abcam (Cambridge, UK). The anti-HSV-2 ICP8 monoclonal antibody (clone 4E6—H2A024) and the anti-HSV-1/2 gD monoclonal antibody (clone 2C10—HA025) were purchased from Virusys Corporation (Taneytown, MD, USA). The anti-actin antibody was from Millipore (Burlington, MA, USA) (clone C4—MAB1501R). The antibodies peroxidase-conjugated AffiniPure F(ab’)2 Fragment goat anti-mouse IgG (H + L) and goat anti-rabbit IgG (H + L) were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Sureram S., Arduino I., Ueoka R., Rittà M., Francese R., Srivibool R., Darshana D., Piel J., Ruchirawat S., Muratori L., Lembo D., Kittakoop P, & Donalisio M. (2022). The Peptide A-3302-B Isolated from a Marine Bacterium Micromonospora sp. Inhibits HSV-2 Infection by Preventing the Viral Egress from Host Cells. International Journal of Molecular Sciences, 23(2), 947.

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