Anti-Ts-NBLsp antibodies (total IgG, IgG1 and IgG2a) in serum of immunized mice were checked by enzyme-linked immunosorbent assay (ELISA). Briefly, microtitre plates (Corning, Acton, MA) were coated with 100 µL of the recombinant protein (rTs-NBLsp) (50 µg mL−1 in coating buffer, overnight at 4 °C). The plates were washed with PBST and blocked with 5% non-fat dry milk (Sigma, St. Louis, MO) in PBST at 37 °C for 2 h, followed by incubation at 37 °C for 2 h with 100 µL of the serum samples with a 1:50 dilution in PBST with 5% non-fat dry milk. After three washes, the plates were incubated at 37 °C for 2 h with 100 µL well−1 of horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Beijing Dingguo Changsheng Biotechnology Co.Ltd.) at a 1:4000 dilution, or goat anti-mouse IgG1 or goat anti-mouse IgG2a (Abcam, Cambridge, UK) at a 1:500 dilution, which was used for determination of total IgG antibody levels and isotype analysis, respectively. ELISA was developed by chromogen 3,3′,5′,5′-tetramethylbenzidine (TMB, Tiangen Biotech Co., Beijing, China), the reaction was terminated by 2 N H2SO4 solution at 15 min after substrate addition, and the OD 450 (Optical density at 450 nm) value was measured.
Non fat dry milk
Manufactured by Merck Group
Sourced in Germany, United States, United Kingdom
Non-fat dry milk is a powdered form of milk that has been processed to remove the fat content. It is a common ingredient used in various food and beverage products.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Sigmafast o phenylenediamine dihydrochloride, by Merck Group (3 mentions)
96 well microtiter plate, by Thermo Fisher Scientific (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Nonfat dry milk, by Americanbio (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Merck Group (2 mentions)
Goat anti mouse igg hrp, by Merck Group (2 mentions)
Prism v9, by GraphPad (2 mentions)
Enhanced chemiluminescence system, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bicinchoninic acid assay, by Beyotime (2 mentions)
Chemiluminescence substrate, by Bio-Rad (2 mentions)
50 protocols using non fat dry milk
1
Quantitative Antibody Profiling in Mice
2
Western Blotting Analysis of Cellular Proteins
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Western blotting analysis was performed on lysates from tissue or cells in RIPA buffer (Sigma‐Aldrich) supplemented with 10 mM sodium fluoride, 0.5 mM sodium orthovanadate, 1:100 protease inhibitor cocktail (Sigma‐Aldrich), and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein (40 μg) were heat‐denatured in sample‐loading buffer (50 mM Tris–HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), resolved by SDS‐polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes (Protran, nitrocellulose membrane, Amersham). The filters were blocked with Tris‐buffered saline containing 0.05% Tween and 5% non‐fat dry milk (Sigma‐Aldrich) and then incubated overnight with the indicated dilutions of different primary antibodies (Table2 ). All secondary horseradish peroxidase‐conjugated antibodies (BioRad) were diluted 1:5000 in Tris‐buffered saline‐Tween and 2.5% non‐fat dry milk (Sigma‐Aldrich). After incubation with SuperSignal Pico or Femto chemiluminescence substrate (Thermo Scientific), the polypeptide bands were detected with GelDoc chemiluminescence detection system (BioRad). Quantification of relative densitometry was obtained by normalizing vs. background and housekeeping proteins using the QuantityOne software (BioRad).
3
Coimmunoprecipitation of Presenilin-1
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For coimmunoprecipitation experiments, cells were harvested in PBS, centrifuged at 800 g for 10 min, and lysed in 125 mmol/L NaCl, 50 mmol/L Hepes, pH 7.4 (supplemented with 1% Triton X-100 or CHAPS and Complete protease inhibitors) for 30 min at 4 °C. After centrifugation at 16000 g for 15 min, cleared cell extracts were incubated overnight at 4 °C with protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA, United States) and anti-PSN1 or anti-rabbit IgG beads. Immunoprecipitated proteins were resolved on 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Bio-Rad, Richmond, CA, United States). The membranes were blocked for 1 h in PBS containing 0.1% Tween 20 (PBS-T) and 5% non-fat dry milk (Sigma) and reacted with antibodies against PSN1, each diluted 1:1000. The membranes were washed with PBS-T, then incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Sigma), diluted 1:5000, and developed with westernsure ECL substrate (LI-COR Biosciences). Immunoreactive bands were identified by co-migration of prestained protein size markers (Fermentas, Glen Burnie, MD, United States). To confirm equivalent protein loading and transfer, the blots were stripped and re-probed for GAPDH (Santa Cruz Biotechnology).
4
Cisplatin and Chlorogenic Acid Evaluation
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Cisplatin [Onco-Tain®] was purchased from Hospira, UK, LTD. Chlorogenic acid hemihydrate (ab120973) was obtained from Abcam Co. (Cambridge, MA, USA). N-acetylcysteine was purchased from Sigma (St. Louis, MO, USA). Tris buffer, non-fat dry milk, bovine serum albumin, and RIPA buffer were obtained from Sigma (St. Louis, MO, USA). Other chemicals were of the highest grade commercially available.
5
Quantifying SARS-CoV-2 Variant Antibody Titers
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Assays were performed in 96-well microtiter plates (Thermo Fisher) coated with 50 μL of recombinant spike from wild type SARS-CoV-2 or variant viruses B.1.1.7, B.1.351, or B.1.617.2. Plates were incubated at 4°C overnight and then blocked with 200 μL of 3% non-fat dry milk (AmericanBio) in PBS containing 0.1% Tween-20 (PBST) for one hour at room temperature (RT). Sera were serially diluted in 1% non-fat dry milk in PBST and added to the plates. Plates were incubated for 120 min at room temperature and then washed 3 times with PBST. Goat anti-mouse IgG-HRP (Sigma-Aldrich, 1:9000) was diluted in 1% non-fat dry milk in PBST before adding to the wells and incubating for 60 min at room temperature. Plates were washed 3 times with PBST before the addition of peroxidase substrate (SigmaFAST o-phenylenediamine dihydrochloride, Sigma-Aldrich). Reactions were stopped by the addition of 3 M hydrochloric acid. Optical density (OD) measurements were taken at 490 nm, and endpoint titers were calculated in excel using a 0.15 OD 490 nm cutoff. Graphs were generated using Graphpad Prism v9.
6
Western Blot Protein Analysis
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Protein Lysates were mixed with SDS-PAGE sample buffer (CST, #7723) and run on 4–20% gradient tris-glycine gels (Life Technologies). Proteins were transferred to nitrocellulose (Millipore, Billerica, MA) and blocked for 1h in 5% nonfat dry milk (Sigma) in TBST. Primary antibodies were incubated in 5% BSA in TBST overnight at 5 °C. Membranes were washed 4 times (15 min each) with TBST, incubated with anti-rabbit or anti-mouse secondary antibody (CST #5366 and #5470) for 1 h at room temperature in 5% milk TBST, washed 3 times (5 min each) with TBST, and developed on ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA).
7
Western Blot Analysis of Retina Proteins
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Adult retinas and other tissue samples were lysed in radioimmunoprecipitation assay buffer consisting of 50-mM Tris (pH 7.4), 150-mM NaCl, 1-mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, and protease inhibitors. Transiently transfected HEK293 cells were lysed using 1× Laemmli buffer. Samples were electrophoresed in a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene difluoride (PVDF) western blotting membrane (Roche Diagnostics), which was blocked with 5% non-fat dry milk and 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) in PBS containing 0.1% Tween 20 (Sigma-Aldrich). Primary antibodies were incubated overnight at 4°C followed by incubation with the corresponding secondary antibodies for 1 hour at room temperature using standard procedures: rabbit αCERKL2 (1:1000, in-house), rabbit αCERKL5 (1:1000, in-house), mouse αGFAP (1:500; MAB8360, MilliporeSigma, Burlington, MA, USA), mouse αGAPDH (1:1000; ab8245, Abcam, Cambridge, UK), horseradish peroxidase (HRP)-conjugated α-mouse (1:2000; ab5706, Abcam), Rabbit IgG HRP Linked Whole Ab (1:2000, NA934VS; GE HealthCare, Chicago, IL, USA).
8
Western Blot Analysis of Proteins from VICs
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Cell extracts from VICs were boiled for 10 minutes and proteins were loaded onto polyacrylamide gels followed by electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with TBS 1X/0.1% Tween-20 containing 5% non-fat dry milk (Sigma-Aldrich, ON, Canada), incubated with antibodies against CCN2 (#E-AB-12339, Elabscience, TX, USA), NELF-E (#10705-1-AP, Proteintech, IL, USA), COL1A1 (#84336, Cell Signaling Technology, New England Biolabs, ON, Canada) or CALNEXIN (#2679, Cell Signaling Technology, New England Biolabs, ON, Canada) overnight at 4°C. Membranes were then washed and incubated with HRP-labeled secondary antibodies (TransBionovo Co., Civic Biosciences Ltd, QC, Canada). Detection was done using clarity western ECL substrate (BioRad, ON, Canada). Images were acquired and quantification analyses were performed using a ChemiDocMP (BioRad, ON, Canada) system and the ImageJ software.
9
Western Blot Analysis of TLR4 Expression
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Western blotting was conducted as described previously (3 (link),12 (link)). Briefly, the membranes were blocked for 30 min at room temperature with 5% non-fat dry milk (Sigma-Aldrich, St. Louis, MO, USA) and immunoblotted using anti-TLR4 (cat. no. sc-293072; monoclonal mouse anti-rat; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following several washes, the membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse IgG (cat. no. sc-2005; 1:2,000; Santa Cruz Biotechnology, Inc.) to detect TLR4 expression. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. no. sc-47724; monoclonal mouse anti-rat; 1:1,000; Santa Cruz Biotechnology, Inc.) and its corresponding secondary antibody, HRP-conjugated polyclonal goat anti-mouse IgG (1:2,000; Santa Cruz Biotechnology, Inc.) served as a control. Protein-antibody complexes were detected with an enhanced chemiluminescence system (Keygen Biotech Co., Ltd.). Protein band sizes were estimated using AlphaView 2.2.14407 software (ProteinSimple, Santa Clara, CA, USA). The density was correlated to the protein expression and normalized to GAPDH.
10
Western Blot Analysis of Protein Expression
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Proteins were extracted from the drug-treated cells under different experimental conditions. Equivalent amounts of sample proteins were separated by 8–12% SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% non-fat dry milk (Sigma-Aldrich) in TBS-T (10 mM Tris-HCl, PH8.0, 150 mM NaCl, and 0.5% Tween 20) for 2 h at room temperature and incubated with anti-caspases-3 (Abcam Inc., Cambridge, UK;1:500), anti-CRABP2 (Proteintech, Chicago, IL, USA; 1:200), anti-DNMT1 (Bioss. Inc., Beijing, China; 1:300), anti-DNMT3A (Bioss. Inc., Beijing, China; 1:1,000), and anti-DNMT3B (Bioss. Inc., Beijing, China; 1:300) primary antibodies overnight at 4°C. After three washes with TBS-T, the membrane was incubated for 1.5 h with HRP-conjugated anti-mouse or anti-rabbit IgG (Zymed Lab, Inc.). Protein expression was detected using chemiluminescent ECL reagents (Roche GmbH, Mannheim, Germany). A Gel-Pro analyzer was used to measure the density of bands. Simultaneously, the expression of a target protein was normalized to that of β-Actin.
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