After mice were killed with with CO2 asphyxiation, eyes from 9-month-old Efemp1+/+, Efemp1ki/ki, and Efemp1−/− mice were enucleated, and the lens and anterior segments were removed. The retina was detached using forceps, the eyecup filled with PBS buffer, and the RPE was removed using a fine camel's hair brush. The BrM/Ch was detached from the sclera, weighed, and minced. Five sets of BrM/Ch samples from five mice per genotype (Efemp1+/+, Efemp1ki/ki, or Efemp1−/−) were analyzed. Samples were digested with 300 μg papain (Sigma-Aldrich Corp.) in 20 mM sodium phosphate buffer (pH 6.8) containing 1 mM EDTA and 2 mM dithiothreitol at 60°C for 2 hours. Samples were diluted with 10 mM iodoacetic acid and 50 mM Tris/HCl (pH 8.0). The sGAG content in papain-digested samples was measured by colorimetric assay with DMMB using an assay kit (Amsbio, Cambridge, MA, USA) according to the manufacturer's instructions. sGAG measurements from Efemp1ki/ki or Efemp1−/− mice were compared with those in Efemp1+/+ mice using a Student's t-test.
Papain
Manufactured by AMS Biotechnology
Sourced in United States
Papain is a proteolytic enzyme derived from the papaya plant. It functions as a catalyst to break down proteins into smaller peptides and amino acids.
Automatically generated - may contain errors
2 protocols using papain
1
Bruch's Membrane Glycosaminoglycan Measurement
2
Isolation and Characterization of Vascular Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents were purchased from Sigma Aldrich unless otherwise noted. Cell culture media was obtained from ThermoFisher Scientific and Lipidure®-CM from AMS Biotechnology (Europe) Ltd. The enzymes used for cell isolation were collagenase Type F, collagenase Type 3 (Worthington, NJ, USA), papain (Worthington) and hyaluronidase. Cell culture dishes with glass coverslip bases (Ibidi µ-Dish 35 mm, high) were purchased from Thistle Scientific (UK). Tali™ Apoptosis Kit (Annexin V Alexa Fluor™ 488) and Dil-conjugated oxLDL from Human Plasma (Dil-OxLDL) were both from Invitrogen™ (ThermoFisher Scientific). The antibodies used for immunocytochemistry were mouse anti-SMA-Cy3 (C6198, Sigma-Aldrich), rabbit anti-Galectin 3 (PA579595 ThermoFisher Scientific) and goat anti-rabbit-AlexaFluor633 (A21071, ThermoFisher Scientific), along with Hoechst33342 Solution (ThermoFisher Scientific).
The buffers used during cell isolation were: Mops buffer (145mM sodium chloride, 2mM MOPS, 4.7mM potassium chloride, 1.2 mM monosodium phosphate, 5mM glucose, 0.02 mM EDTA, 2mM sodium pyruvate, 1.2mM magnesium chloride, 2mM calcium chloride, pH 7.4) and isolation buffer (80mM sodium glutamate, 55mM sodium chloride, 6mM potassium chloride, 10mM glucose, 10mM Hepes,1mM magnesium chloride, 0.1mM calcium chloride, 0.2mM EDTA, pH 7.4), with or without 2 mg/ml fatty acid free bovine serum albumin (BSA).
The buffers used during cell isolation were: Mops buffer (145mM sodium chloride, 2mM MOPS, 4.7mM potassium chloride, 1.2 mM monosodium phosphate, 5mM glucose, 0.02 mM EDTA, 2mM sodium pyruvate, 1.2mM magnesium chloride, 2mM calcium chloride, pH 7.4) and isolation buffer (80mM sodium glutamate, 55mM sodium chloride, 6mM potassium chloride, 10mM glucose, 10mM Hepes,1mM magnesium chloride, 0.1mM calcium chloride, 0.2mM EDTA, pH 7.4), with or without 2 mg/ml fatty acid free bovine serum albumin (BSA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!