Flow channels (constructed of easy-cleaned [25 (link)] glass coverslips and silicon wafers with an oxide layer of 30nm) were flushed with a sequence of: (i) casein solution consisting of 0.5mg/ml casein (Sigma) in BRB80 in order to block the flow-channel surfaces (incubation time 5min), (ii) motor solution consisting of 150nM kinesin-1, 1mM MgATP (Roche), 0.2mg/ml casein, 10mM DDT (Sigma) in BRB80 in order to bind kinesin-1 proteins unspecifically to the casein surface (incubation time 5min), (iii) S-MT solution consisting of S-MTs diluted in BRB80T supplemented with 1mM MgATP, 0.2mg/ml casein, 10mM DDT (incubation time 2 min) and (iv) imaging solution consisting of 1mM MgATP, 0.2mg/ml casein, 10mM DDT, and an oxygen scavenger mixture (consisting of 40mM glucose [Sigma], 110μg/ml glucose oxidase [SERVA], 22μg/ml catalase [Sigma]) in BRB80T to remove the excess MTs. For experiments using QD-SA-B-S-MTs instead of S-MTs, 10-20pM streptavidin conjugated QDot 655 (Lifetechnologies) solution was added to the B-S-MT solution and after an incubation time of 2 min this mixture was diluted and used as stated in (iii).
Casein
Manufactured by Merck Group
Sourced in United States, Germany, Spain, Australia, United Kingdom, China
Casein is a type of milk protein that is commonly used as a laboratory reagent. It is a complex mixture of related phosphoproteins that are the main proteins in milk. Casein serves as a core function as a source of amino acids and other nutrients for cell growth and maintenance in various cell culture and biochemical applications.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (27 mentions)
Trypsin, by Merck Group (10 mentions)
Tween 20, by Merck Group (9 mentions)
Sodium carbonate, by Merck Group (9 mentions)
Gelatin, by Merck Group (9 mentions)
Tris hcl, by Merck Group (8 mentions)
Yeast extract, by Merck Group (8 mentions)
Trichloroacetic acid, by Merck Group (8 mentions)
Sucrose, by Merck Group (8 mentions)
Azocasein, by Merck Group (7 mentions)
Hydrochloric acid (hcl), by Merck Group (7 mentions)
Methanol, by Merck Group (6 mentions)
Perchloric acid, by Merck Group (6 mentions)
Gallic acid, by Merck Group (6 mentions)
Starch, by Merck Group (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Glucose, by Merck Group (6 mentions)
Ethanol, by Merck Group (5 mentions)
Sodium hydroxide (naoh), by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
273 protocols using casein
1
Flow Channel Assembly for Kinesin-MT Assay
2
Casein-PR Agar Media for Metabolism
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The casein-PR agar media used for metabolism testing contained 1% casein (Sigma-Aldrich, 22090), 0.5% NaCl (S3014), 0.15% Bacto Yeast extract (BD, 212750), 0.008% Phenol red (PR, Sigma-Aldrich, P4758), and 1.3% agar. For additional supplements, 1% sucrose (Sigma-Aldrich, S7903) or 1% lactose (Sigma, 61345) was added to the casein-PR media. Experiments were performed using the same methods for the AST studies.
3
Cardosin B Digestion of Casein Proteins
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Purified cardosin B (11 g/mL) was added to pure -casein, -casein and -casein (Sigma,USA) (200 g/mL) in 100 mM sodium phosphate buffer pH 6.4 in a ratio 1:100 (v/v) and incubated at 37 °C. Time-points 0, 5, 15, 30, 60, 120 and 180 min were collected and analyzed. Control for non-enzymatic digestion was performed using 20 mM Tris-HCl pH 7.4 instead of cardosin B and was collected at 180 min.
4
Porcine Trypsin Casein Hydrolysis Assay with COOH-f-MWCNTs
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The porcine trypsin (Merck, Darmstadt, Germany) was dissolved in 20 mM Tris (2-Amino-2-(hydroxymethyl)-1, 3-propanediol, Trometamol, THAM, Tris base) buffer, pH 8.0 to form a 5.0 × 10−5 M-1 solution. The casein solution (1% (w/v)): 0/5 g of casein (Merck, Darmstadt, Germany) was suspended in 40 ml of Tris buffer and was entirely dissolved by placing on a water bath for 20 min. The solution was cooled and made up to 50 ml with Tris buffer. Trichloroacetic acid (TCA 10% (w/v)): 5 g TCA (Sigma, St. Louis, MO, USA) was dissolved in 50 ml dH2O. COOH-f-MWCNTs (OD: 20–30 nm, Length: 10–30 μm, SSA: >110 m2/g, Content of -COOH: 2.73 wt % were dissolved in ultrapure water (based on the TEM image of S1 Fig ) using an urtra-sonicator prior to the experiment. Purity Multi-walled nanotubes>98wt %) were obtained from Iranian Nanomaterials Pioneers, Mashhad, Iran. Tris was purchased from Sigma (St. Louis, MO, USA).
5
Quantifying Chicken Immunoglobulins by ELISA
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Immunoglobulins (IgA and IgY) were quantified in samples from both animal trials by enzyme-linked immunosorbent assay (ELISA) according to Kothlow et al. (61 (link)). Briefly, 96-well plates were coated overnight with 2 μg/ml or 2.5 μg/ml murine monoclonal antibodies (mABs) in coating buffer (pH 9.6) for IgA (A1, SouthernBiotech, Birmingham, AL, USA) and IgY (G1, SouthernBiotech, Birmingham, AL, USA), respectively. Plates were blocked in 1% casein (Merck KGaA, Darmstadt, Germany) in PBS (pH 7.4) followed by subsequent incubations at room temperature with serial dilutions of chicken plasma, bile, or cecal content samples. Horseradish peroxidase (HRP)-coupled mABs against chicken IgA (A3-HRP) and IgY (G1-HRP) were used at a concentration of 1:10,000 in 1% casein in PBS-T (pH 7.4) followed by detection with tetramethylbenzidine (Merck KGaA, Darmstadt, Germany). Gibco chicken serum (Thermo Fisher Scientific, Waltham, MA, USA) was used as a standard control (IgA: 81 μg/ml; IgY: 1,685 μg/ml) to quantify immunoglobulins.
6
Motility Assay of Microtubules
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Motility assays were performed as previously described57 (link). Rhodamine-labelled microtubules (Cytoskeleton) were polymerized in BRB80 buffer (80 mM PIPES, 1 mM MgCl2 and 1 mM EGTA) containing 1 mM GTP at 37 °C for 10 min. Flow chambers with a volume of ∼10 μl were constructed with adhesive tapes on glass slides and incubated with 1 mg ml−1 casein (Sigma) and kinesin (50 μM for Kid and 20 μM for K560). MTs were diluted in BRB80 buffer with 0.5 mg ml−1 casein, 22.5 mM glucose, 0.22 mg ml−1 glucose oxidase, 0.036 mg ml−1 catalase, 3 mM ATP and 10 μM taxol (Sigma), and were introduced into the flow chamber. MTs were incubated in the presence or absence of 20 nM NuSAP protein at room temperature for 5 min before flowing into the chamber. Unbound microtubules were washed off and images were collected near the middle of the flow cell using the Ultraview Vox Spinning disc confocal system (PerkinElmer).
7
In Vitro Protein Digestibility Analysis
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The in vitro protein digestibility analysis was conducted according to the methods described by Kumagai et al. with some modifications. (25) (link) As a reference, casein (Sigma-Aldrich) was used. For RA, WA, or casein, 100 mg was suspended in 10 mL of distilled water, adjusted to pH 2 with dilute HC1, and incubated at 37 °C for 2 h together with 1 mg of porcine pepsin (Sigma-Aldrich). After incubation, the pepsin was inactivated by neutralization with 100 mg of NaHCO 3 . Subsequently, 10 mg of porcine pancreatin (Sigma-Aldrich) was added and incubated at 37 °C for 2, 4, and 6 h. Then, 50 μL of each solution was collected and mixed with 950 μL of an SDS-sample buffer. These samples were used for SDS-PAGE analysis with 14% acrylamide gel, which was conducted according to the methods described by Laemmli. (26) (link) The gel was stained with Coomassie Brilliant Blue (CBB).
8
Colloidal Gold Conjugation with BLV Antigen
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Colloidal gold was prepared as previously described [11 ], with minor modifications. Briefly, 1 mL of a 1% solution of chloroauric acid (Sigma) was added to 100 mL of distilled water and boiled. While boiling, 2.4 mL of a 1% solution of trisodium citrate dihydrate was added to the solution. The gold solution gradually formed as the citrate reduced the gold (III). When the solution began to develop a deep red color, heating was discontinued. Colloidal gold (40 nm) (Median Diagnostics) and BLV lysate antigen were prepared as previously described [28 (link)] and added drop-wise into the colloidal gold solution (final concentration 10 µg/mL) over a 1 h period at room temperature while being stirred. Casein (Sigma) was added as a blocking agent (final concentration at 0.3%), and the mixture was stirred for 1 h at room temperature. After the mixture was centrifuged at 14,000 × g and 4℃ for 30 min, the supernatant was discarded and the resulting pellet was resuspended in 1/10 of its original volume in 2 mM borax buffer containing 1% BSA. Following removal of the colloidal gold-recombinant antigen conjugate to a 1.5 mL tube, 5% sucrose (Sigma), 0.1% Casein, and 0.1% sodium azide (Sigma) were added, and the concentration of the conjugate was adjusted to OD450 = 1.5. Following these steps, the conjugate solution was allowed to dry.
9
ELISA for Screening VHH-Fc Antibodies
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For ELISAs with VHH-Fcs, recombinant SARS-CoV-2 spike glycoprotein ectodomains were coated overnight at 4 °C onto NUNC Immulon 4 HBX microtiter plates (Thermo Fisher, Cat# 3855) at 50 ng/well in 100 µL of phosphate-buffered saline (PBS), pH 7.4. The next day, plates were blocked with 200 µl PBSC (1% (w/v) casein (Sigma, Cat# E3414) in PBS) for 1 h at room temperature, then washed five times with PBST (PBS supplemented with 0.05% (v/v) Tween 20) and incubated at room temperature for 1 h on rocking platform at 80 rpm with 100 µl of various concentrations of VHH-Fcs diluted in PBSTC (PBS/0.2% casein/0.1% Tween 20). Plates were washed five times with PBSTC and binding of VHH-Fcs was detected using 100 µl of 1 µg/ml horse radish peroxidase-conjugated goat anti-human IgG (Sigma, Cat# A0170). Finally, wells were washed 10 times and incubated with 100 µl peroxidase substrate solution (SeraCare, Cat# 50-76-00) at room temperature for 15 min. Reactions were stopped by adding 50 µl of 1 M H2SO4 to wells, and absorbance were subsequently measured at 450 nm using a Multiskan FC photometer (Thermo Fisher). The same steps were performed for ELISAs with monomeric VHHs, except that biotinylated VHHs were used instead of VHH-Fcs and the binding of the VHHs was detected using horseradish peroxidase-conjugated-streptavidin (Jackson ImmunoResearch, Cat# 016-030-084).
10
Erythropoietin and Casein Effects on Rats
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The animal experiments described in the present work were all gained approval of the Animal Ethics Committee of Chongqing Medical University and the Animal Management Rule of the Chinese Ministry of Health. Eight-week-old male SD rats were bought from the Animal Experimental Center of Chongqing Medical University. All rats were provided with high phosphorus (1.2%) diets under conditions of 12-h light–dark cycles at 23°C. Followed one-week adaptive feeding, the laboratory animals were classified into four groups randomly (10 of each): control group, EPO group, casein group, and EPO+casein group. High-dose EPO 2000 U/Kg/W (Life-iLab, Shanghai, China) was employed for injections intraperitoneally to rats in both EPO and EPO+casein groups three times per week, lasting for 20 consecutive weeks. Simultaneously, 10% casein (1.2 g/kg) (Sigma, USA) was subcutaneously injected into the rats in casein group and the EPO+casein group every other day, also lasting for 20 weeks [25 (link)]. The control group was administered normal saline (Southwest Pharmaceutical Co., Ltd.) at an identical quantity. Ultimately, all rats were put to death to collect the abdominal aortic vessels for subsequent experiments.
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