CS was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). GM, genipin, β-TCP, and boric acid were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Gelatin was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Hematoxylin and eosin were purchased from Sigma Aldrich Trading Co., Ltd. (Shanghai, China). Methyl methacrylate was purchased from Shanghai Zhanyun Chemical Co. Ltd. (Shanghai, China). Paraformaldehyde was purchased from Shanghai Lingfeng Chemical Reagent Co. Ltd. (Shanghai, China). Acetic and hydrochloric acids were purchased from Xilong Scientific Co., Ltd. (Guangdong, China).
Gelatin
Manufactured by Solarbio
Sourced in China
Gelatin is a protein-based material derived from the partial hydrolysis of collagen. It is a translucent, colorless, and flavorless substance that is commonly used in various laboratory applications. Gelatin possesses the ability to form a gel-like substance when dissolved in hot water and subsequently cooled.
Automatically generated - may contain errors
Lab products found in correlation
Genipin, by Maclin Biochemical Technology (1 mentions)
Boric acid, by Maclin Biochemical Technology (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
β tcp, by Maclin Biochemical Technology (1 mentions)
Paraformaldehyde (pfa), by Shanghai Lingfeng Chemical Reagent (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Collagen, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
4 protocols using gelatin
1
Biomaterial Synthesis and Characterization
2
Gelatin-coated Coverslips for Cell Culture
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Before cell culture, the sterile coverslips were coated with 1% Gelatin (Solarbio, G8061) in the 24-well plate in the cell culture hood for at least 1 h. Then discard the excess liquid and dry the coverslips completely in the cell culture hood. The cavity and blood cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, 11965092), supplemented with 10% fetal bovine serum (Gibco, 10099141) at 37 °C in the presence of 5% CO2. After incubation at 37 °C for 4–6 h, removed the cell culture medium and rinsed the cells 3 times with PBS. The following procedures were carried out using standard procedures.
3
Evaluating Gelatinase Activity in LAB
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The gelatinase activity of LAB was conducted by using a previously reported method with minor adjustments (Perin et al., 2014 (link); Rastogi et al., 2020 (link)). Briefly, 1 μL of 24 h incubated LAB was spotted on MRS agar with 5% (w/v) gelatin (Solarbio, China), and the plates were incubated anaerobically at 37°C for 72 h, then cooled at 4°C for 4 h. The opaque halo around the colony is considered to be a positive result of gelatinase production.
4
Hydrolytic Activity of Keratinase BsKER71
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To determine the hydrolytic reaction of selected natural proteins, 5 μL of gelatin (Solarbio, Beijing, China), fibrin (Solarbio, Beijing, China), collagen (Sigma Chemical Co., St. Louis, USA), BSA (Solarbio, Beijing, China), or casein (10 mg/mL) (AOBOX, Beijing, China) was mixed with 5 μL (4.8 μg) of purified BsKER71 in 20 μL boric acid–NaOH buffer (pH 9.6). After incubation for 20 min at 50 °C, proteolytic reaction was terminated by heating at 95 °C for 10 min. Then, samples were directly loaded onto 12% SDS-PAGE.
The keratinase activity was determined as previously described using keratin powder (XABC biotech Co., Xian, China) as substrate (Wawrzkiewicz et al. 1987 (link)). One unit of keratinolytic activity was defined as the amount of enzyme required to generate an increase of 0.01 OD value at 280 nm.
Similarly, caseinolytic activity was determined using previously described method (Wan et al. 2009 (link)). Folin–Ciocalteau reagent was purchased from Sangon Co. (Shanghai, China). One unit of caseinolytic activity was defined as the amount of enzyme required to produce 1 μg of tyrosine per min.
The keratinase activity was determined as previously described using keratin powder (XABC biotech Co., Xian, China) as substrate (Wawrzkiewicz et al. 1987 (link)). One unit of keratinolytic activity was defined as the amount of enzyme required to generate an increase of 0.01 OD value at 280 nm.
Similarly, caseinolytic activity was determined using previously described method (Wan et al. 2009 (link)). Folin–Ciocalteau reagent was purchased from Sangon Co. (Shanghai, China). One unit of caseinolytic activity was defined as the amount of enzyme required to produce 1 μg of tyrosine per min.
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