Nonfat dry milk

Manufactured by Southern Biotech

Nonfat dry milk is a powdered form of milk that has had the fat and water content removed. It is a common ingredient used in various laboratory applications, serving as a stabilizer, emulsifier, and protein source.

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Lab products found in correlation

Non fat dry milk, by Bio-Rad (3 mentions) Tmb substrate, by R&D Systems (2 mentions) Medisorp plates, by Merck Group (2 mentions) Tween 20, by Merck Group (2 mentions) Infinite m1000, by Tecan (2 mentions) Hrp conjugated goat anti mouse igg1 igg2a, by Southern Biotech (1 mentions) Tween 20, by Bio-Rad (1 mentions) Tween 20, by Southern Biotech (1 mentions) Goat serum, by Thermo Fisher Scientific (1 mentions) Goat serum, by Southern Biotech (1 mentions) Phosphatase substrate tablets, by Merck Group (1 mentions) Dpbs t, by Merck Group (1 mentions)

3 protocols using nonfat dry milk

ELISA for Mouse Antibody Responses

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Flat bottom 384‐well MediSorp plates (Sigma‐Aldrich) were coated with 0.4 μg/mL (20 µL/well) of HEK293T‐produced E1E2 antigen in PBS and incubated overnight at 4 °C. After extensive washing with PBS containing 0.05% Tween 20 (Sigma‐Aldrich), plates were blocked for 1 h, at RT with PBS containing 5% nonfat dry milk (BioRad) and then washed as before. Six binary dilutions, starting from 1:200, of serum in PBS containing 5% nonfat dry milk were added to the antigen‐coated plates (40 μL/well) and incubated for 2 h, at RT. The plates were washed, incubated with HRP‐conjugated goat anti‐mouse IgM/IgG (Southern Biotech, AL), diluted 6000‐fold or with HRP‐conjugated goat anti‐mouse IgG1/IgG2a (Southern Biotech) diluted 1000‐fold in PBS containing 5% nonfat dry milk, for 2 h, at RT. Enzymatic reactions were developed following the addition of the TMB substrate (R&D Systems), for 20 min at RT then stopped with 20 μL/well of 1 m H₂SO₄. Absorbance was measured at 450 nm using a Tecan Infinite M1000 microplate reader. Normalization across ELISA plates was performed by pooling all the immune sera from day 49 which served as an internal standard. Serum pool dilution starting from 1:100 was used as an internal standard on each plate and a 4‐parameter logistic regression model was fitted to the measured values.

Dobrica M., van Eerde A., Tucureanu C., Onu A., Paruch L., Caras I., Vlase E., Steen H., Haugslien S., Alonzi D., Zitzmann N., Bock R., Dubuisson J., Popescu C., Stavaru C., Liu Clarke J, & Branza‐Nichita N. (2021). Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus‐neutralizing activity in vaccinated mice. Plant Biotechnology Journal, 19(10), 2027-2039.

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ELISA for Anti-HCV Antibody Detection

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Flat‐bottom 384‐well MediSorp plates (Sigma‐Aldrich, St.Louis, USA) were coated with 0.24 μg/mL of HEK293T cell‐produced E1E2 antigen, in PBS, overnight at 4 °C. The plates were washed five times with PBS containing 0.05% Tween 20 (Sigma‐Aldrich, St.Louis, USA), blocked with PBS containing 5% nonfat dry milk (Bio‐Rad, Hercules, USA), for 1 h at room temperature and further washed 5 times with PBS containing 0.05% Tween 20. Serum samples were twofold serially diluted (1:200–1:6400) in PBS containing 5% nonfat dry milk then added to the antigen coated plates (40 μL/well) and incubated for 2 h at room temperature. Faecal suspensions (40 μL/well) were twofold serially diluted (1 : 2–1 : 8) as above. The plates were washed five times with PBS containing 0.05% Tween 20, before incubation with HRP‐conjugated goat anti‐mouse IgM/IgG/IgA adsorbed against human immunoglobulins (Southern Biotech), diluted 6000‐fold in PBS containing 5% nonfat dry milk, for 2 h at room temperature. The faeces samples were incubated with HRP‐conjugated goat anti‐mouse IgA only. The plates were washed five times before addition of the TMB substrate (R&D Systems) and further incubated for 20 minutes at room temperature. Reactions were stopped with 20 μL/well of 1M H₂SO₄ and absorbance was measured at 450 nm using a Tecan Infinite M1000 microplate reader.

Clarke J.L., Paruch L., Dobrica M., Caras I., Tucureanu C., Onu A., Ciulean S., Stavaru C., Eerde A., Wang Y., Steen H., Haugslien S., Petrareanu C., Lazar C., Popescu C., Bock R., Dubuisson J, & Branza‐Nichita N. (2017). Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination. Plant Biotechnology Journal, 15(12), 1611-1621.

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EEEV E2 Glycoprotein ELISA Protocol

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Recombinant EEEV E2 glycoprotein (E3E2) (strain V105; IBT Bioservices) was diluted to 0.5 μg/mL in 1x D-PBS to coat 384-well ELISA plates (Thermo Fisher Scientific) at 25 μL/well and incubated at 4°C overnight. The plates were washed 3x with D-PBS-T (1x D-PBS + 0.05% Tween 20 [Cell Signaling Technology]) and blocked for 1 hour at room temperature with 25 μL/well blocking solution (2% non-fat dry milk (Bio-Rad), 2% goat serum (Gibco) in D-PBS-T). After blocking, the plates then were washed 3x with D-PBS-T and a volume of 10 to 25 μL/well of supernatant from each well containing EBV-transformed B cells or hybridoma cell lines was added. Plates were incubated for 2 hours at room temperature or overnight at 4°C. Plates then were washed 3x with D-PBS-T and a suspension of secondary antibodies (goat anti-human IgG-AP (Meridian Life Science) and goat anti-human IgA-AP [Southern Biotech]) at a 1:4,000 dilution in 1% blocking solution (1% non-fat dry milk, 1% goat serum) was added at 25 μL/well for 1 hour at room temperature. Alkaline phosphatase substrate solution (phosphatase substrate tablets (Sigma-Aldrich) in AP substrate buffer (1M Tris aminomethane and 30 mM MgCl₂) was added at 25 μL/well following plate washing 4x with D-PBS-T. Plates were incubated at room temperature in the dark for 1–2 hours and then read at an optical density of 405 nm with a plate reader.

Williamson L.E., Gilliland T J.r., Yadav P., Binshtein E., Bombardi R., Kose N., Nargi R.S., Sutton R.E., Durie C.L., Armstrong E., Carnahan R.H., Walker L.M., Kim A.S., Fox J.M., Diamond M.S., Ohi M.D., Klimstra W.B, & Crowe JE J.r. (2020). Human antibodies protect against aerosolized Eastern equine encephalitis virus infection. Cell, 183(7), 1884-1900.e23.

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