PTs endocytic capacity of Ocrl and Clcn5 mice was examined by measuring β-lactoglobulin and dextran uptake. β-lactoglobulin was tagged with Cy5 using TM2 Ab labeling kit (Amersham) in accordance with the manufacturer’s instructions. Fifteen minutes after tail-vein injection of Cy5-β-lactoglobulin (1 mg/kg B.W., L3908, Sigma) or 30 min after injection of 10 kDa Alexa 647-dextran (6 mg/kg B.W.; D22914, Thermo Fisher Scientific), mice were anesthetized and their kidneys were harvested and processed for confocal microscopy. The endocytic capacity of Ocrl and Clcn5 mPTCs was examined by measuring albumin and dextran uptake as described previously (44 (link)). Briefly mPTCs were incubated at 37°C with 100 μg ml−1 Alexa488-BSA (A13100, Thermo Fisher Scientific) or 250 μg ml−1 Alexa 647-dextran diluted in medium without FBS supplementation, for 15 and 30 min, respectively. After washing, the cells were fixed in 4% PFA and processed for confocal microscopy.
L3908
Manufactured by Merck Group
Sourced in Germany
The L3908 is a laboratory instrument designed for precision measurement and analysis. It is capable of performing a variety of tasks within a controlled laboratory environment. The core function of the L3908 is to provide accurate and reliable data collection, without interpretation or extrapolation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
D22914, by Thermo Fisher Scientific (2 mentions)
H9250, by Merck Group (1 mentions)
A1887, by Merck Group (1 mentions)
A83 01, by Merck Group (1 mentions)
A2153, by Merck Group (1 mentions)
Cholecalciferol, by Merck Group (1 mentions)
C9756, by Merck Group (1 mentions)
L6010, by Merck Group (1 mentions)
7 protocols using l3908
1
Endocytic Capacity in Ocrl and Clcn5 Mice
2
Receptor-Mediated Endocytosis in Mouse Kidneys
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Receptor-mediated endocytosis was monitored in the PT segments of mouse kidneys by measuring the uptake of β-lactoglobulin (L3908, Sigma). Briefly, β-lactoglobulin was tagged with Cy5 using TM2 Ab labelling kit (Amersham) following the manufacturer’s instructions. Fifteen minutes after tail-vein injection of Cy5-β-lactoglobulin (1 mg/kg B.W.) mice were anesthetized and their kidneys were harvested and processed by confocal microscopy.
3
Protein and Enzyme Characterization Protocol
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Proteins and enzymes were obtained from Sigma-Aldrich (St. Louis, MO, USA)/ Merck (Darmstadt, Germany) and were used as received. All solutions were prepared in ultrapure water in phosphate buffer 10 mM pH 7.2. Water was purified with the Hydrolab SPRING 20UV system. Proteins and enzymes were dissolved in PBS (phosphate-buffered saline) solution to appropriate concentrations immediately before measurements. Final concentrations of the solutions were verified spectrophotometrically using the molar absorption coefficients: ε280nm = 35,600 M−1 cm−1 for ovalbumin (Sigma-Aldrich, A5503), ε280nm = 53,500 M−1 cm−1 for papain (Roth, 8933.1), ε280nm = 18,000 M−1 cm−1 for β-lactoglobulin (Sigma-Aldrich, L3908), ε280nm = 39,000 M−1 cm−1 for lysozyme (Sigma-Aldrich, 62970), and ε280nm = 5800 M−1 cm−1 for insulin (Sigma-Aldrich, 91077C). The histone from calf thymus was obtained from Sigma-Aldrich (H9250).
4
Measuring Ctns Rat PT Endocytic Capacity
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The endocytic capacity of Ctns rat PTs in vivo was examined by measuring β-lactoglobulin and dextran uptake. β-Lactoglobulin was tagged with Cy5 using a TM2 Ab labeling kit (GERPN4000, GE Healthcare, Chicago, IL, USA) in accordance with the manufacturer’s instructions. Twenty minutes after tail-vein injection of Cy5-β-lactoglobulin (0.4 mg kg−1 B.W., L3908, Sigma-Aldrich) or 30 min after injection of 10 kDa Alexa 647-dextran (0.2 mg kg−1 B.W.; D22914, Thermo Fisher Scientific), rats were anesthetized and their kidneys were harvested and processed for confocal microscopy. The endocytic uptake of Ctns rPTCs was monitored using Cy5-β-lactoglobulin. The cells were incubated with the indicated concentrations of Cy5-β-lactoglobulin, diluted in FBS-deprived culture medium for 20 min at 37°C (36 ).
5
Isoelectric Profiling of Albumin Variants
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To further compare the UP with commercial human albumin (A1887, Sigma) and commercial glycated human albumin (A8301, Sigma), the iso-electro point of the three proteins was determined (Figure 1C ). Equal amounts (6.9 μg) of the three proteins were run along with standards on a precast Bio-Rad IEF gel pH 3–10 (Bio-Rad, Hercules, CA, United States). The IEF standards were soybean trypsin inhibitor, 4.6 pI (10109886001, Sigma), and bovine milk β-lactoglobulin A, 5.1 pI (L3908, Sigma). The protein bands of interest were flanked on both sides by the standards in the gel. The gel was run for 3 h at various voltages; 1 h at 100 V, 1 h at 250 V, and 30 min at 500 V. The gel was fixed in 10% trichloroacetic acid (TCA) for 10 min. Excess ampholytes were removed by an additional overnight 1% TCA soak. To detect the protein bands, the gel was washed in water three times and stained with GELCODE Blue Stain Reagent (Pierce, Rockville, IL, United States).
6
Protein Incubation and Cell Fixation
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Cells were placed on ice and washed with cold PBS, followed by an incubation with 0.25 mg/mL albumin (A2153, Sigma Aldrich, Taufkirchen, Germany) or with 0.25 mg/mL ß-lactoglobulin (L3908, Sigma Aldrich, Taufkirchen, Germany) at 37 °C for 5 min or 15 min, respectively. Afterwards, cells were placed on ice, washed five times with cold PBS, and fixed in 4% PFA/PBS for 10 min.
7
Calcium-depleted Milk Protein Fortification
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Calcium-depleted α-LA from bovine milk (≥85% pure, L6010), β-LG (≥90% pure, L3908), and cholecalciferol (vitD, ≥98%, C9756) were from Sigma-Aldrich (St. Louis, MO). All other reagents were of high purity or HPLC grade.
To keep conditions close to those that vitD is likely to encounter in fortified drinks, we used low concentrations (5 mM) of the following buffers in combination with 15 mM NaCl: glycine (pH 2-3), acetate (pH 4-5), and Tris (pH 7.4). pH was adjusted with HCl or NaOH.
To keep conditions close to those that vitD is likely to encounter in fortified drinks, we used low concentrations (5 mM) of the following buffers in combination with 15 mM NaCl: glycine (pH 2-3), acetate (pH 4-5), and Tris (pH 7.4). pH was adjusted with HCl or NaOH.
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