Structured Review

FUJIFILM proteinase k
R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4  (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.
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Images

1) Product Images from "Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities"

Article Title: Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv116

R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4  (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.
Figure Legend Snippet: R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.

Techniques Used: Incubation, Concentration Assay, SDS Page, Polyacrylamide Gel Electrophoresis

R.PabI restricted DNA without strand breaks. ( A ) Design. Double-stranded, circular plasmid DNA was treated with R.PabI and transformation activity is measured. Box, recognition sequence. Me, base methylation within the recognition sequence. Only one recognition sequence is shown for simplicity. ( B ) R.PabI treatment at 37°C does not cause single-strand breaks. Plasmid pBAD30_ cviQIM (0.11 pmol, 11 nM, Supplementary Figure S1) purified from E. coli under noninducing conditions was incubated with or without R.PabI (0.77 pmol, 77 nM) at 37°C for 30 min, which was followed by proteinase K (ProK) treatment at 37°C overnight to inactivate R.PabI. DNA was incubated at 85°C for 6 h. Alternatively, plasmid was incubated with or without R.PabI at 85°C for 6 h. Samples were subjected to 0.8% agarose gel electrophoresis. OC, open circle; SC, supercoiled circle; P, product DNA. Left lane: 1 kb DNA ladder. ( C ) Loss of DNA transformation after R.PabI treatment. pBAD30_ cviQIM (0.11 pmol, 11 nM, Supplementary Figure S1) with varying levels of methylation was treated with R.PabI (0.77 pmol, 77 nM) at 37°C for 30 min and purified for quantitative transformation (‘Materials and Methods’ section, Supplementary Figure S5) of E. coli . The plasmid was prepared from cultures with 0.2% arabinose overnight, 0.2% arabinose 5 h, 0.02% arabinose 5 h, 0.002% arabinose 5 h, 0.0002% arabinose 5 h, and glucose overnight, in the order of expected decreased methylation level. E. coli HST08 was transformed with R.PabI-treated plasmids, and resulting colonies were counted to determine transformation efficiencies.
Figure Legend Snippet: R.PabI restricted DNA without strand breaks. ( A ) Design. Double-stranded, circular plasmid DNA was treated with R.PabI and transformation activity is measured. Box, recognition sequence. Me, base methylation within the recognition sequence. Only one recognition sequence is shown for simplicity. ( B ) R.PabI treatment at 37°C does not cause single-strand breaks. Plasmid pBAD30_ cviQIM (0.11 pmol, 11 nM, Supplementary Figure S1) purified from E. coli under noninducing conditions was incubated with or without R.PabI (0.77 pmol, 77 nM) at 37°C for 30 min, which was followed by proteinase K (ProK) treatment at 37°C overnight to inactivate R.PabI. DNA was incubated at 85°C for 6 h. Alternatively, plasmid was incubated with or without R.PabI at 85°C for 6 h. Samples were subjected to 0.8% agarose gel electrophoresis. OC, open circle; SC, supercoiled circle; P, product DNA. Left lane: 1 kb DNA ladder. ( C ) Loss of DNA transformation after R.PabI treatment. pBAD30_ cviQIM (0.11 pmol, 11 nM, Supplementary Figure S1) with varying levels of methylation was treated with R.PabI (0.77 pmol, 77 nM) at 37°C for 30 min and purified for quantitative transformation (‘Materials and Methods’ section, Supplementary Figure S5) of E. coli . The plasmid was prepared from cultures with 0.2% arabinose overnight, 0.2% arabinose 5 h, 0.02% arabinose 5 h, 0.002% arabinose 5 h, 0.0002% arabinose 5 h, and glucose overnight, in the order of expected decreased methylation level. E. coli HST08 was transformed with R.PabI-treated plasmids, and resulting colonies were counted to determine transformation efficiencies.

Techniques Used: Plasmid Preparation, Transformation Assay, Activity Assay, Sequencing, Methylation, Purification, Incubation, Agarose Gel Electrophoresis

2) Product Images from "Renal papillary tip extract stimulates BNP production and excretion from cardiomyocytes"

Article Title: Renal papillary tip extract stimulates BNP production and excretion from cardiomyocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0197078

Characterization of papillary extracts by proteolysis and molecular weight fractionation. A, Papillary extracts were digested with 0.3 μg/ml Proteinase K for each indicated time and loaded into 10% and 15% gel. The pictures are representative of three experiments. B, BNP mRNA expression was examined by Northern blot analysis in cardiomyocytes treated with control buffer (MOCK) or each digested extract of the papillary tip. *P
Figure Legend Snippet: Characterization of papillary extracts by proteolysis and molecular weight fractionation. A, Papillary extracts were digested with 0.3 μg/ml Proteinase K for each indicated time and loaded into 10% and 15% gel. The pictures are representative of three experiments. B, BNP mRNA expression was examined by Northern blot analysis in cardiomyocytes treated with control buffer (MOCK) or each digested extract of the papillary tip. *P

Techniques Used: Molecular Weight, Fractionation, Expressing, Northern Blot

3) Product Images from "Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution"

Article Title: Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution

Journal: BMC Research Notes

doi: 10.1186/s13104-018-3914-4

The effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed K1 cells. RNA was extracted from filter-trapped K1 cells after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C
Figure Legend Snippet: The effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed K1 cells. RNA was extracted from filter-trapped K1 cells after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C

Techniques Used: Incubation

Effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed thyroid FNAB specimens. The thyroid FNAB specimens processed for LBC using CytoRich-Red solution were trapped to filters in the same way as described above. RNA was extracted from the specimens after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C
Figure Legend Snippet: Effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed thyroid FNAB specimens. The thyroid FNAB specimens processed for LBC using CytoRich-Red solution were trapped to filters in the same way as described above. RNA was extracted from the specimens after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C

Techniques Used: Incubation

RNA recovery from filter-trapped K1 cells fixed for liquid-based cytology. RNA was extracted from the fixed K1 cells after incubation with Proteinase K-containing for the indicated periods at 55 °C. RNA recovery from K1 cells fixed with CytoRich-Red ( a ) and CytoRich-Blue ( b ). Black bars and gray bars represent the RNA yield from 20,000 to 100,000 cells, respectively. Quality of RNAs extracted from K1 cells fixed with CytoRich-Red ( c ) and CytoRich-Blue ( d ). The analysis using Agilent 2100 bioanalyzer revealed that 28S and 18S ribosomal RNAs (correspond to the bands at approximately 1900 nt and 3900 nt on the charts, respectively) remained nearly intact, leading to high RINs.  N/A  not assessed
Figure Legend Snippet: RNA recovery from filter-trapped K1 cells fixed for liquid-based cytology. RNA was extracted from the fixed K1 cells after incubation with Proteinase K-containing for the indicated periods at 55 °C. RNA recovery from K1 cells fixed with CytoRich-Red ( a ) and CytoRich-Blue ( b ). Black bars and gray bars represent the RNA yield from 20,000 to 100,000 cells, respectively. Quality of RNAs extracted from K1 cells fixed with CytoRich-Red ( c ) and CytoRich-Blue ( d ). The analysis using Agilent 2100 bioanalyzer revealed that 28S and 18S ribosomal RNAs (correspond to the bands at approximately 1900 nt and 3900 nt on the charts, respectively) remained nearly intact, leading to high RINs. N/A not assessed

Techniques Used: Incubation

4) Product Images from "Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution"

Article Title: Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution

Journal: BMC Research Notes

doi: 10.1186/s13104-018-3914-4

The effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed K1 cells. RNA was extracted from filter-trapped K1 cells after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C
Figure Legend Snippet: The effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed K1 cells. RNA was extracted from filter-trapped K1 cells after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C

Techniques Used: Incubation

Effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed thyroid FNAB specimens. The thyroid FNAB specimens processed for LBC using CytoRich-Red solution were trapped to filters in the same way as described above. RNA was extracted from the specimens after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C
Figure Legend Snippet: Effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed thyroid FNAB specimens. The thyroid FNAB specimens processed for LBC using CytoRich-Red solution were trapped to filters in the same way as described above. RNA was extracted from the specimens after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C

Techniques Used: Incubation

RNA recovery from filter-trapped K1 cells fixed for liquid-based cytology. RNA was extracted from the fixed K1 cells after incubation with Proteinase K-containing for the indicated periods at 55 °C. RNA recovery from K1 cells fixed with CytoRich-Red ( a ) and CytoRich-Blue ( b ). Black bars and gray bars represent the RNA yield from 20,000 to 100,000 cells, respectively. Quality of RNAs extracted from K1 cells fixed with CytoRich-Red ( c ) and CytoRich-Blue ( d ). The analysis using Agilent 2100 bioanalyzer revealed that 28S and 18S ribosomal RNAs (correspond to the bands at approximately 1900 nt and 3900 nt on the charts, respectively) remained nearly intact, leading to high RINs.  N/A  not assessed
Figure Legend Snippet: RNA recovery from filter-trapped K1 cells fixed for liquid-based cytology. RNA was extracted from the fixed K1 cells after incubation with Proteinase K-containing for the indicated periods at 55 °C. RNA recovery from K1 cells fixed with CytoRich-Red ( a ) and CytoRich-Blue ( b ). Black bars and gray bars represent the RNA yield from 20,000 to 100,000 cells, respectively. Quality of RNAs extracted from K1 cells fixed with CytoRich-Red ( c ) and CytoRich-Blue ( d ). The analysis using Agilent 2100 bioanalyzer revealed that 28S and 18S ribosomal RNAs (correspond to the bands at approximately 1900 nt and 3900 nt on the charts, respectively) remained nearly intact, leading to high RINs. N/A not assessed

Techniques Used: Incubation

5) Product Images from "Anti-inflammatory effect of aqueous extract from Kawachi-bankan (Citrus maxima) peel in vitro and in vivo"

Article Title: Anti-inflammatory effect of aqueous extract from Kawachi-bankan (Citrus maxima) peel in vitro and in vivo

Journal: Cytotechnology

doi: 10.1007/s10616-019-00323-4

Properties of the active substance in Kawachi-bankan peel aqueous extract (KPE). a To investigate the heat stability of the active substance in KPE, 10 mg/mL of KPE was heated at 100 °C for each time (20, 40, and 60 min). b To investigate the susceptibility of the active substance in KPE to proteases, 10 mg/mL of KPE was treated with various concentrations of proteinase K for at 37 °C for 20 h and subsequently heated at 100 °C for 5 min to inactivate the protease. RAW264.7 cells were treated with 10% FBS-DMEM containing 100 ng/mL of LPS and treated with KPE or 10 mM NaPB and incubated for 12 h. After incubation, the concentration of IL-6 in the culture media was measured by ELISA. Data are representative of three independent experiments performed in triplicate and expressed as the mean ± standard deviation. n.s., no statistical significance by Tukey–Kramer test
Figure Legend Snippet: Properties of the active substance in Kawachi-bankan peel aqueous extract (KPE). a To investigate the heat stability of the active substance in KPE, 10 mg/mL of KPE was heated at 100 °C for each time (20, 40, and 60 min). b To investigate the susceptibility of the active substance in KPE to proteases, 10 mg/mL of KPE was treated with various concentrations of proteinase K for at 37 °C for 20 h and subsequently heated at 100 °C for 5 min to inactivate the protease. RAW264.7 cells were treated with 10% FBS-DMEM containing 100 ng/mL of LPS and treated with KPE or 10 mM NaPB and incubated for 12 h. After incubation, the concentration of IL-6 in the culture media was measured by ELISA. Data are representative of three independent experiments performed in triplicate and expressed as the mean ± standard deviation. n.s., no statistical significance by Tukey–Kramer test

Techniques Used: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Related Articles

Mobility Shift:

Article Title: Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes the enhanced fast velocity of APP transport by kinesin-1
Article Snippet: The antibody was purified from serum by serial affinity chromatography with the antigen and then by passage through resin conjugated with nonphosphorylated KLC1462–470 peptide (C+TVTTTLKNL-CONH2 ), and a resin conjugated with phosphothreonine (Sigma-Aldrich). .. Phos-tag–based mobility shift assay FLAG-KLC1 expressed in COS7 cells was recovered by immunoprecipitation with anti-FLAG M2 antibody and separated by Phos-tag SDS–PAGE (Wako Pure Chemical Industries, Osaka, Japan) ( ). ..

Immunoprecipitation:

Article Title: Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes the enhanced fast velocity of APP transport by kinesin-1
Article Snippet: The antibody was purified from serum by serial affinity chromatography with the antigen and then by passage through resin conjugated with nonphosphorylated KLC1462–470 peptide (C+TVTTTLKNL-CONH2 ), and a resin conjugated with phosphothreonine (Sigma-Aldrich). .. Phos-tag–based mobility shift assay FLAG-KLC1 expressed in COS7 cells was recovered by immunoprecipitation with anti-FLAG M2 antibody and separated by Phos-tag SDS–PAGE (Wako Pure Chemical Industries, Osaka, Japan) ( ). ..

SDS Page:

Article Title: Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes the enhanced fast velocity of APP transport by kinesin-1
Article Snippet: The antibody was purified from serum by serial affinity chromatography with the antigen and then by passage through resin conjugated with nonphosphorylated KLC1462–470 peptide (C+TVTTTLKNL-CONH2 ), and a resin conjugated with phosphothreonine (Sigma-Aldrich). .. Phos-tag–based mobility shift assay FLAG-KLC1 expressed in COS7 cells was recovered by immunoprecipitation with anti-FLAG M2 antibody and separated by Phos-tag SDS–PAGE (Wako Pure Chemical Industries, Osaka, Japan) ( ). ..

Incubation:

Article Title: Growth differentiation factor-11 supplementation improves survival and promotes recovery after ischemic stroke in aged mice
Article Snippet: .. Tissue sections were incubated for 1 hour in blocking solution (0.1% Triton-X, 10% normal goat serum in 1X PBS) and then incubated overnight in primary antibody at 4°C (GDF11/8 1:250; IBA-1(Fujifilm Wako pure chemical corporation, NCNP24) 1:300; GFAP-Cy3 (Sigma-Aldrich C9205) 1:300; CD 31 (ab28364) 1: 100), DyLight 594 labelled Lycopersicon Esculentum (tomato) lectin (Vector Laboratories)1:100, BDNF (1:100), MBP (1:100, cell signaling technologies, 78896S), synaptophysin (1:300, ab14692). .. BrdU staining Following three washes with 0.1M PBS, pH 7.4 for 10 minutes, sections were placed in ice-cold 1N hydrochloric acid (HCl) for 10 minutes, followed by incubation with 2N HCl for 30 minutes at 370 C. After 30 minutes, the sections were rinsed three times with 0.1M 1X PBS and then sections were incubated with 0.1M boric acid for 10 minutes.

Blocking Assay:

Article Title: Growth differentiation factor-11 supplementation improves survival and promotes recovery after ischemic stroke in aged mice
Article Snippet: .. Tissue sections were incubated for 1 hour in blocking solution (0.1% Triton-X, 10% normal goat serum in 1X PBS) and then incubated overnight in primary antibody at 4°C (GDF11/8 1:250; IBA-1(Fujifilm Wako pure chemical corporation, NCNP24) 1:300; GFAP-Cy3 (Sigma-Aldrich C9205) 1:300; CD 31 (ab28364) 1: 100), DyLight 594 labelled Lycopersicon Esculentum (tomato) lectin (Vector Laboratories)1:100, BDNF (1:100), MBP (1:100, cell signaling technologies, 78896S), synaptophysin (1:300, ab14692). .. BrdU staining Following three washes with 0.1M PBS, pH 7.4 for 10 minutes, sections were placed in ice-cold 1N hydrochloric acid (HCl) for 10 minutes, followed by incubation with 2N HCl for 30 minutes at 370 C. After 30 minutes, the sections were rinsed three times with 0.1M 1X PBS and then sections were incubated with 0.1M boric acid for 10 minutes.

other:

Article Title: Pathogenic Mutations Differentially Regulate Cell-to-Cell Transmission of α-Synuclein
Article Snippet: AntibodiesThe following antibodies were used: anti-α-synuclein monoclonal antibody (BD Transduction Laboratories, San Jose, CA, United States), anti-bovine serum albumin (BSA) monoclonal antibody (Abcam, Cambridge, United Kingdom), anti-β-actin monoclonal antibody (Abcam), anti-α-synuclein (phospho S129) monoclonal antibody (Wako, Tokyo, Japan), anti-tyrosine hydroxylase rabbit antibody (Sigma, Merck KGaA, Darmstadt, Germany), anti-Ibal rabbit antibody (Wako), and anti-C-myc rabbit antibody (Santa Cruz, Dallas, TX, United States).

Recombinant:

Article Title: Cellular and Mathematical Analyses of LUBAC Involvement in T Cell Receptor-Mediated NF-κB Activation Pathway
Article Snippet: For lentiviral transduction, pCSII-CMV-RfA-IRES-Blast (RIKEN BioResource Research Center) was used. .. ReagentsThe following reagents and kits were obtained as indicated: recombinant human TNF-α (BioLegend), phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), ionomycin (Wako), Human IL-2 Instant ELISA (eBiosciences), and NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce). .. AntibodiesThe following antibodies were used for immunoblot analyses: P-p105 (#4806; 1:1,000), p105 (#3035; 1:1,000), P-p65 (#3033; 1:1,000), p65 (#8242; 1:1,000), P-IκBα (#9246; 1:1,000), IκBα (#4812; 1:1,000), P-JNK (#4668; 1:1,000), JNK (#9252; 1:1,000), PARP (#9542; 1:1,000), P-ZAP70 (#2701; 1:1,000), ZAP70 (#3165; 1:2,000), P-IKKα/β (#2697; 1:1,000), CARMA1 (#4435; 1:1,000), MALT1 (#2494; 1:1,000), BCL10 (#4237; 1:1,000), CYLD (#8462; 1:1,000), OTULIN (#14127; 1:1,000), and GST (#2622; 1:1,000) were obtained from Cell Signaling.

Enzyme-linked Immunosorbent Assay:

Article Title: Cellular and Mathematical Analyses of LUBAC Involvement in T Cell Receptor-Mediated NF-κB Activation Pathway
Article Snippet: For lentiviral transduction, pCSII-CMV-RfA-IRES-Blast (RIKEN BioResource Research Center) was used. .. ReagentsThe following reagents and kits were obtained as indicated: recombinant human TNF-α (BioLegend), phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), ionomycin (Wako), Human IL-2 Instant ELISA (eBiosciences), and NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce). .. AntibodiesThe following antibodies were used for immunoblot analyses: P-p105 (#4806; 1:1,000), p105 (#3035; 1:1,000), P-p65 (#3033; 1:1,000), p65 (#8242; 1:1,000), P-IκBα (#9246; 1:1,000), IκBα (#4812; 1:1,000), P-JNK (#4668; 1:1,000), JNK (#9252; 1:1,000), PARP (#9542; 1:1,000), P-ZAP70 (#2701; 1:1,000), ZAP70 (#3165; 1:2,000), P-IKKα/β (#2697; 1:1,000), CARMA1 (#4435; 1:1,000), MALT1 (#2494; 1:1,000), BCL10 (#4237; 1:1,000), CYLD (#8462; 1:1,000), OTULIN (#14127; 1:1,000), and GST (#2622; 1:1,000) were obtained from Cell Signaling.

Article Title: Impact of Acipimox Therapy on Free Fatty Acid Efflux and Endothelial Function in the Metabolic Syndrome: A Randomized Trial
Article Snippet: .. Plasma insulin was measured using an Immunoenzymatic Sandwich Assay (Beckman Coulter, Inc., Brea, CA); plasma FFA concentration using an in vitro enzymatic colorimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) with reagents from Wako Chemicals (Richmond, VA); high-sensitivity C-reactive protein (hsCRP) using an immunoturbidimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) using reagents and calibrators from DiaSorin (Stillwater, MN); tumor necrosis factor (TNF) receptor 2 (TNFR2) using ELISA (R & D Systems, Minneapolis, MN); and myeloperixodase (MPO) using ELISA (Alpco Diagnostics, Salem, NH). ..

Concentration Assay:

Article Title: Impact of Acipimox Therapy on Free Fatty Acid Efflux and Endothelial Function in the Metabolic Syndrome: A Randomized Trial
Article Snippet: .. Plasma insulin was measured using an Immunoenzymatic Sandwich Assay (Beckman Coulter, Inc., Brea, CA); plasma FFA concentration using an in vitro enzymatic colorimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) with reagents from Wako Chemicals (Richmond, VA); high-sensitivity C-reactive protein (hsCRP) using an immunoturbidimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) using reagents and calibrators from DiaSorin (Stillwater, MN); tumor necrosis factor (TNF) receptor 2 (TNFR2) using ELISA (R & D Systems, Minneapolis, MN); and myeloperixodase (MPO) using ELISA (Alpco Diagnostics, Salem, NH). ..

In Vitro:

Article Title: Impact of Acipimox Therapy on Free Fatty Acid Efflux and Endothelial Function in the Metabolic Syndrome: A Randomized Trial
Article Snippet: .. Plasma insulin was measured using an Immunoenzymatic Sandwich Assay (Beckman Coulter, Inc., Brea, CA); plasma FFA concentration using an in vitro enzymatic colorimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) with reagents from Wako Chemicals (Richmond, VA); high-sensitivity C-reactive protein (hsCRP) using an immunoturbidimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) using reagents and calibrators from DiaSorin (Stillwater, MN); tumor necrosis factor (TNF) receptor 2 (TNFR2) using ELISA (R & D Systems, Minneapolis, MN); and myeloperixodase (MPO) using ELISA (Alpco Diagnostics, Salem, NH). ..

Enzymatic Colorimetric Assay:

Article Title: Impact of Acipimox Therapy on Free Fatty Acid Efflux and Endothelial Function in the Metabolic Syndrome: A Randomized Trial
Article Snippet: .. Plasma insulin was measured using an Immunoenzymatic Sandwich Assay (Beckman Coulter, Inc., Brea, CA); plasma FFA concentration using an in vitro enzymatic colorimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) with reagents from Wako Chemicals (Richmond, VA); high-sensitivity C-reactive protein (hsCRP) using an immunoturbidimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) using reagents and calibrators from DiaSorin (Stillwater, MN); tumor necrosis factor (TNF) receptor 2 (TNFR2) using ELISA (R & D Systems, Minneapolis, MN); and myeloperixodase (MPO) using ELISA (Alpco Diagnostics, Salem, NH). ..

Immunoturbidimetry Assay:

Article Title: Impact of Acipimox Therapy on Free Fatty Acid Efflux and Endothelial Function in the Metabolic Syndrome: A Randomized Trial
Article Snippet: .. Plasma insulin was measured using an Immunoenzymatic Sandwich Assay (Beckman Coulter, Inc., Brea, CA); plasma FFA concentration using an in vitro enzymatic colorimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) with reagents from Wako Chemicals (Richmond, VA); high-sensitivity C-reactive protein (hsCRP) using an immunoturbidimetric assay on a Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) using reagents and calibrators from DiaSorin (Stillwater, MN); tumor necrosis factor (TNF) receptor 2 (TNFR2) using ELISA (R & D Systems, Minneapolis, MN); and myeloperixodase (MPO) using ELISA (Alpco Diagnostics, Salem, NH). ..

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    FUJIFILM proteinase k
    Properties of the active substance in Kawachi-bankan peel aqueous extract (KPE). a To investigate the heat stability of the active substance in KPE, 10 mg/mL of KPE was heated at 100 °C for each time (20, 40, and 60 min). b To investigate the susceptibility of the active substance in KPE to proteases, 10 mg/mL of KPE was treated with various concentrations of <t>proteinase</t> K for at 37 °C for 20 h and subsequently heated at 100 °C for 5 min to inactivate the protease. RAW264.7 cells were treated with 10% FBS-DMEM containing 100 ng/mL of LPS and treated with KPE or 10 mM NaPB and incubated for 12 h. After incubation, the concentration of IL-6 in the culture media was measured by ELISA. Data are representative of three independent experiments performed in triplicate and expressed as the mean ± standard deviation. n.s., no statistical significance by Tukey–Kramer test
    Proteinase K, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase k/product/FUJIFILM
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    Properties of the active substance in Kawachi-bankan peel aqueous extract (KPE). a To investigate the heat stability of the active substance in KPE, 10 mg/mL of KPE was heated at 100 °C for each time (20, 40, and 60 min). b To investigate the susceptibility of the active substance in KPE to proteases, 10 mg/mL of KPE was treated with various concentrations of proteinase K for at 37 °C for 20 h and subsequently heated at 100 °C for 5 min to inactivate the protease. RAW264.7 cells were treated with 10% FBS-DMEM containing 100 ng/mL of LPS and treated with KPE or 10 mM NaPB and incubated for 12 h. After incubation, the concentration of IL-6 in the culture media was measured by ELISA. Data are representative of three independent experiments performed in triplicate and expressed as the mean ± standard deviation. n.s., no statistical significance by Tukey–Kramer test

    Journal: Cytotechnology

    Article Title: Anti-inflammatory effect of aqueous extract from Kawachi-bankan (Citrus maxima) peel in vitro and in vivo

    doi: 10.1007/s10616-019-00323-4

    Figure Lengend Snippet: Properties of the active substance in Kawachi-bankan peel aqueous extract (KPE). a To investigate the heat stability of the active substance in KPE, 10 mg/mL of KPE was heated at 100 °C for each time (20, 40, and 60 min). b To investigate the susceptibility of the active substance in KPE to proteases, 10 mg/mL of KPE was treated with various concentrations of proteinase K for at 37 °C for 20 h and subsequently heated at 100 °C for 5 min to inactivate the protease. RAW264.7 cells were treated with 10% FBS-DMEM containing 100 ng/mL of LPS and treated with KPE or 10 mM NaPB and incubated for 12 h. After incubation, the concentration of IL-6 in the culture media was measured by ELISA. Data are representative of three independent experiments performed in triplicate and expressed as the mean ± standard deviation. n.s., no statistical significance by Tukey–Kramer test

    Article Snippet: To investigate the susceptibility of the active substance in KPE to proteases, 10 mg/mL of KPE was treated with various concentrations of proteinase K (Fujifilm Wako Pure Chemical) at 37 °C for 20 h and subsequently heated at 100 °C for 5 min to inactivate the protease.

    Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation