Native BoNT/C and BoNT/A1 were purified as previously described [66 (link), 67 (link)]. Cytosine β-D-arabinofuranoside hydrochloride (C6645), DNAse I from bovine pancreas (DN25), poly-L-lysine hydrobromide (P1274) and trypsin (T4799) were from Sigma Aldrich. μ-Conotoxin GIIIB is from Alomone, Jerusalem, Israel. Primary antibodies: anti-SNAP-25 (SMI81, ab24737) was from Abcam. Anti-SNAP-25 (cleaved) and syntaxin-1A/1B polyclonal antibodies were produced in our laboratory and previously characterized [37 (link), 68 (link)]. Secondary antibodies conjugated to HRP were from Calbiochem; secondary antibodies for immunofluorescence conjugated to Alexa Fluorophores 488 or 555 and α-Bungarotoxin conjugated to Alexa 647 were from Thermo Scientific, Waltham, MA, USA.
Trypsin t4799
Manufactured by Merck Group
Sourced in United States
Trypsin (T4799) is a digestive enzyme derived from bovine pancreas. It is commonly used in cell culture applications to facilitate the dissociation and detachment of adherent cells from cell culture surfaces.
Automatically generated - may contain errors
Lab products found in correlation
Cytosine β d arabinofuranoside hydrochloride c6645, by Merck Group (2 mentions)
Poly l lysine hydrobromide p1274, by Merck Group (1 mentions)
μ conotoxin giiib, by Alomone (1 mentions)
Curcumin, by Merck Group (1 mentions)
Anti vamp2, by Synaptic Systems (1 mentions)
Spectrostar nano microplate reader, by BMG Labtech (1 mentions)
Cell strainer, by Corning (1 mentions)
Laminin l2020, by Merck Group (1 mentions)
Fetal bovine serum f7524, by Merck Group (1 mentions)
Ab1670, by Abcam (1 mentions)
Ab75810, by Abcam (1 mentions)
Ab4680, by Abcam (1 mentions)
Amd3100, by Abcam (1 mentions)
Conotoxin giiib, by Alomone (1 mentions)
5 protocols using trypsin t4799
1
Purification and Characterization of BoNT/C and BoNT/A1
2
Neuronal SNARE Protein Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytosine β-D-arabinofuranoside hydrochloride (C6645), DNase I from bovine pancreas (DN25), poly-L-lysine hydrobromide (P1274) and trypsin (T4799) were purchased from Sigma Aldrich (St. Louis, Missouri, USA).
Primary antibodies: anti-SNAP-25 (SMI81, ab24737) was purchased from Abcam(Cambridge, UK), while anti-syntaxin-1A (STX-1A, 110111) and anti-VAMP-2 were purchased from Synaptic System (Göttingen, Germany). Secondary antibodies: HRP-conjugated Ab were purchased from Calbiochem® (San Diego, CA, USA), Alexa Fluorophores 488- or 555-conjugated Ab were from Thermo-Fisher Scientific (Waltham, MA, USA). Native TeNT was purified as previously described [58 (link)].
Inhibitors: myricetin [3,3′,4′,5,5′7-Hexahydroxyflavone] and Curcumin [(E,E)-1,7-bis(4-Hydroxy-3- methoxyphenyl)-1,6-heptadiene-3,5-dione] were purchased from Sigma Aldrich. PX12 [2-[(1-Methylpropyl) dithio]-1H-imidazole] and Ebselen [2-Phenyl-1,2-benzisoselenazol3(2H)-one] were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 4-Bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone (EGA) was synthesized as in [47 (link)].
Primary antibodies: anti-SNAP-25 (SMI81, ab24737) was purchased from Abcam(Cambridge, UK), while anti-syntaxin-1A (STX-1A, 110111) and anti-VAMP-2 were purchased from Synaptic System (Göttingen, Germany). Secondary antibodies: HRP-conjugated Ab were purchased from Calbiochem® (San Diego, CA, USA), Alexa Fluorophores 488- or 555-conjugated Ab were from Thermo-Fisher Scientific (Waltham, MA, USA). Native TeNT was purified as previously described [58 (link)].
Inhibitors: myricetin [3,3′,4′,5,5′7-Hexahydroxyflavone] and Curcumin [(E,E)-1,7-bis(4-Hydroxy-3- methoxyphenyl)-1,6-heptadiene-3,5-dione] were purchased from Sigma Aldrich. PX12 [2-[(1-Methylpropyl) dithio]-1H-imidazole] and Ebselen [2-Phenyl-1,2-benzisoselenazol3(2H)-one] were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 4-Bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone (EGA) was synthesized as in [47 (link)].
3
Inhibitory Activity of TaSRTRG Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TaSRTRG inhibitory activities against trypsin T4799 (Sigma-Aldrich, France) and chymotrypsin C4129 (Sigma-Aldrich, France) were determined by incubating purified TaSRTRG proteins together with the enzymes and measuring the change in absorbance at 25°C for 30 min. The remaining trypsin activity was measured with TAME T4626 (Sigma-Aldrich, France) as a substrate and the changes in absorbance at 247 nm were measured with a UV-Visible spectrometer SPECTROstar Nano Microplate Reader (BMG LABTECH, Germany) in a 0.3 ml reaction mixture containing 46 mM Tris/HCl (pH 8.1), 11.5 mM CaCl2 and 1 mM TAME. For the measurement of a-chymotrypsin activity, BTEE B6125 (Sigma-Aldrich, France) was used and the absorbency at 256 nm was followed by a 0.3 ml reaction mixture containing 40 mM Tris/HCl (pH 7.8), 50 mM CaCl2 and 0.5 mM BTEE. Commercial soybean BBI T9777 (Sigma-Aldrich, France) was assayed in parallel to serve as a positive control. Three independent assays were conducted per substrate per protein in each of three trials (n = 9 per SSP per substrate).
4
Isolation and Culture of Myoblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The myoblasts were isolated and cultured according to the protocol described by Fauconneau and Paboeuf (2000) [103 (link)]. The fast-twitch muscles were collected from the epaxial region and mechanically dissociated with scalpels. To release the muscle cells, the fragments were enzymatically digested with 0.2% collagenase type I (C9891) and 0.1% trypsin (T4799) (Sigma-Aldrich, St. Louis, MO, USA). The cell suspension was filtered in cell strainers (Corning, New York, NY, USA), allowing for the removal of debris, centrifuged, and the cell pellet was resuspended in DMEM medium (DMEM (D7777), 9 mM NaHCO3 (S5761), 20 mM HEPES (H3375), pH 7.4), with 1% antibiotics (A5955) and 10% fetal bovine serum (F7524) (Sigma-Aldrich, St. Louis, MO, USA). The cells were diluted at a concentration of 2 × 106 cells/mL and plated in 6-wells plates, previously treated with poly-L-lysine (P6282) and laminin (L2020) (Sigma-Aldrich, St. Louis, MO, USA), which have high affinity for the myoblasts. The myoblasts were incubated at 28 °C, with the medium changed every day, and the myoblasts morphology was monitored regularly under a microscope (Olympus, Tokyo, Japan). The results were achieved from 3 independent cell cultures.
5
NUCC-390 Synthesis and Neuronal Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NUCC‐390 synthesis was performed as previously described.22 AMD3100, a strong CXCR4 antagonist, was from Abcam (120718). Cytosine β‐D‐arabinofuranoside (C6645), DNase I (DN25), poly‐L‐lysine (P1274), laminin (L2020), and trypsin (T4799) were from Sigma Aldrich. µ‐conotoxin GIIIB was from Alomone. Primary antibodies: β3‐tubulin (302302, Synaptic System), NF (ab4680, Abcam), CXCR4 (ab1670, Abcam), GAP43 (ab75810, Abcam), CXCL12α (Cell Signaling, BK3740S). Secondary antibodies were from Thermo Scientific.
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!