Proteasome activity assays were performed as described previously (Devulapalli et al., 2019 (link); McFadden et al., 2019 ; Orsi et al., 2019 (link)). Normalized samples (nuclear = 2μg; cytoplasmic = 20μg) were diluted in MilliQ H2O and mixed with reaction buffer (250mM HEPES, pH 7.5, 5mM EDTA, 0.5% NP-40, 0.01% SDS, 5mM ATP). Fluorogenic peptide Suc-leu-leu-val-thy-AMC (#BML-P802-0005, Enzo Life Sciences) was then added to the samples at a concentration of 10μM to assess proteasome chymotrypsin-like activity. The reaction was incubated at 37°C for 2 hrs and fluorescence monitored at 360 (excitation)/460 (emission) on a monochromatic plate reader (Synergy H1; Biotek, Winooski, VT). Protein free blanks were used and an AMC standard curve was produced. The scan with the peak level of AMC was used for statistical analyses. Data are presented as the percentage change in relative fluorescent units (RFU) relative to the Control group.
Suc leu leu val thy amc
Manufactured by Enzo Life Sciences
Suc-leu-leu-val-thy-AMC is a fluorogenic substrate that can be used to detect and measure the activity of the protease enzyme cathepsin B. It is a peptide sequence (succinyl-leucine-leucine-valine-tyrosine-7-amino-4-methylcoumarin) that is cleaved by cathepsin B, releasing the fluorescent 7-amino-4-methylcoumarin (AMC) compound which can be detected and quantified.
Automatically generated - may contain errors
Lab products found in correlation
Synergy h1, by Agilent Technologies (5 mentions)
Bml p802 0005, by Enzo Life Sciences (1 mentions)
Bz val gly arg amc, by Enzo Life Sciences (1 mentions)
Z leu leu glu amc, by Enzo Life Sciences (1 mentions)
Bml bw9375 0005, by Enzo Life Sciences (1 mentions)
Bml zw9345 0005, by Enzo Life Sciences (1 mentions)
5 protocols using suc leu leu val thy amc
1
Proteasome Activity Assay Protocol
2
Proteasome Chymotrypsin-like Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Proteasome activity assays were performed as described previously (Jarome et al. 2021 (link)). Normalized samples (15 µg) were diluted with MilliQ H2O and mixed with reaction buffer (250 mM HEPES at pH 7.5, 5 mM EDTA, 0.5% NP-40, 0.01% SDS, 5 mM ATP). Fluorogenic peptide Suc-leu-leu-val-thy-AMC (Enzo Life Sciences BML-P802-0005) at a concentration of 10 µM was then added to each sample to assess proteasome chymotrypsin-like activity. Using a monochromatic plate reader (Synergy H1, Biotek), the reaction was incubated for 2 h at 37°C and fluorescence-monitored at 360 (excitation)/460 (emission). Protein-free blanks were used to generate an AMC standard curve. The scan with the peak level of AMC was used for statistical analyses. Data are presented as the percentage change in relative fluorescent units (RFUs) relative to the Scr-siRNA group.
3
Proteasome Activity Assay Protocol
4
Proteasome Chymotrypsin-like Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteasome activity assays were performed as described previously (Devulapalli et al., 2019 (link); McFadden et al., 2019 (link); Orsi et al., 2019 (link)) Normalized samples (10 μg) were diluted in MilliQ H2O and mixed with reaction buffer (250 mM HEPES, pH 7.5, 5 mM EDTA, 0.5% NP-40, 0.01% SDS, 5 mM ATP). Fluorogenic peptide Suc-leu-leu-val-thy-AMC (#BML-P802-0005, Enzo Life Sciences) was then added to the samples at a concentration of 10 μM to assess proteasome chymotrypsin-like activity. The reaction was incubated at 37°C for 2 h and fluorescence monitored at 360 (excitation)/460 (emission) on a monochromatic plate reader (Synergy H1; Biotek, Winooski, VT, USA). Protein-free blanks were used and an AMC standard curve was produced. The scan with the peak level of AMC was used for statistical analyses. Data are presented as the percentage change in relative fluorescent units (RFU) relative to the Control group.
5
Proteasome Activity Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteasome activity assays were performed as described previously (Jarome et al. 2013 (link); Devulapalli et al. 2019 (link); Orsi et al. 2019 (link)) with minor modifications. Cytoplasmic and synaptic fractions were normalized to 10 and 5 µg/µL, respectively. Samples were diluted in dH2O and mixed with reaction buffer (500 mM HEPES at pH 7.5, 500 mM EDTA, 10% NP-40, 10% SDS, 10 mM ATP). The fluorogenic peptide Suc-leu-leu-val-thy-AMC (Enzo Life Sciences BML-P802-0005) was added to samples to assess chymotrypsin-like activity (10 µM). The reaction was incubated for 2 h at 37°C, and fluorescence was monitored at 360 (excitation)/460 (emission) on a monochromatic plate reader (Synergy H1, Biotek). Protein-free blanks were used, and an AMC standard curve was produced. The scan with the peak level of AMC was used for statistical analyses. Data are presented as the percent change in relative fluorescent units (RFUs) relative to the naïve group.
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