Structured Review

Fisher Scientific proteinase k
Mechanism and physico-chemical model of enzymatic micromachining. (a) Microchannels with spatially varying cross-sectional profiles are constructed by co-injection of an enzyme (proteinase K, PK) and an inhibitor (bovine serum albumin, BSA) through a
Proteinase K, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Characterization of enzymatic micromachining for construction of variable cross-section microchannel topologies"

Article Title: Characterization of enzymatic micromachining for construction of variable cross-section microchannel topologies

Journal: Biomicrofluidics

doi: 10.1063/1.4948508

Mechanism and physico-chemical model of enzymatic micromachining. (a) Microchannels with spatially varying cross-sectional profiles are constructed by co-injection of an enzyme (proteinase K, PK) and an inhibitor (bovine serum albumin, BSA) through a
Figure Legend Snippet: Mechanism and physico-chemical model of enzymatic micromachining. (a) Microchannels with spatially varying cross-sectional profiles are constructed by co-injection of an enzyme (proteinase K, PK) and an inhibitor (bovine serum albumin, BSA) through a

Techniques Used: Construct, Injection

2) Product Images from "Genome-Wide Profiling of RNA–Protein Interactions Using CLIP-Seq"

Article Title: Genome-Wide Profiling of RNA–Protein Interactions Using CLIP-Seq

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-3591-8_12

Schematic representation of iCLIP. RNA binding protein (RBP) and RNA are covalently bound in vivo using UV radiation (step 1). Potential RBP–RNA complexes are then purified together (steps 2–5). L3 linker adapter ligation to the 3′ end allows for sequence-specific priming of reverse transcription and the 5′ is radioactively labeled (steps 6 and 7). RBP–RNA complexes are purified from free RNA using SDS-PAGE and wet membrane transfer (step 8). Complexes are recovered from the membrane using Proteinase K (step 9). Reverse transcription truncates at the remaining polypeptide and introduces two cleavable adapter regions and a barcode (step 10). Size selection using Urea-PAGE removes the RT primer prior to circularization of the cDNA (steps 11–13). Linearization generates templates for PCR amplification (steps 14 and 15). High-throughput sequencing generates reads where the barcode sequences are immediately before the last nucleotide of the input cDNA (step 16). This nucleotide is one position upstream of the crosslinked nucleotide, allowing for identification of the RBP–RNA binding site with high resolution
Figure Legend Snippet: Schematic representation of iCLIP. RNA binding protein (RBP) and RNA are covalently bound in vivo using UV radiation (step 1). Potential RBP–RNA complexes are then purified together (steps 2–5). L3 linker adapter ligation to the 3′ end allows for sequence-specific priming of reverse transcription and the 5′ is radioactively labeled (steps 6 and 7). RBP–RNA complexes are purified from free RNA using SDS-PAGE and wet membrane transfer (step 8). Complexes are recovered from the membrane using Proteinase K (step 9). Reverse transcription truncates at the remaining polypeptide and introduces two cleavable adapter regions and a barcode (step 10). Size selection using Urea-PAGE removes the RT primer prior to circularization of the cDNA (steps 11–13). Linearization generates templates for PCR amplification (steps 14 and 15). High-throughput sequencing generates reads where the barcode sequences are immediately before the last nucleotide of the input cDNA (step 16). This nucleotide is one position upstream of the crosslinked nucleotide, allowing for identification of the RBP–RNA binding site with high resolution

Techniques Used: RNA Binding Assay, In Vivo, Purification, Ligation, Sequencing, Labeling, SDS Page, Selection, Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Next-Generation Sequencing

3) Product Images from "In vivo Distribution and Clearance of Purified Capsular Polysaccharide from Burkholderia pseudomallei in a Murine Model"

Article Title: In vivo Distribution and Clearance of Purified Capsular Polysaccharide from Burkholderia pseudomallei in a Murine Model

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0005217

Western blot analysis of excreted CPS. Purified CPS (lane 1) and urine samples (lanes 2–7) were separated on 7.5% SDS-PAGE gels. All samples including purified CPS were incubated with proteinase K at 60°C for 1 hour, followed by boiling for 10 min before loading on the gels. Lanes 2, 3, and 4 were loaded with control urine spiked with CPS and incubated at 37°C for 30 min, 2 hours, and 8 hours, respectively. Lanes 5, 6, and 7 were urine from CPS-injected mice collected at 30 min, 2 hours, and 8 hours post-injection, respectively. The volume of sample loaded into each lane was adjusted to contain an equal amount of CPS, approximately 1 μg/lane. After blotting, membranes were probed with mAb 4C4 (1 μg/mL). Intact CPS was observed in urine samples from CPS-treated mice.
Figure Legend Snippet: Western blot analysis of excreted CPS. Purified CPS (lane 1) and urine samples (lanes 2–7) were separated on 7.5% SDS-PAGE gels. All samples including purified CPS were incubated with proteinase K at 60°C for 1 hour, followed by boiling for 10 min before loading on the gels. Lanes 2, 3, and 4 were loaded with control urine spiked with CPS and incubated at 37°C for 30 min, 2 hours, and 8 hours, respectively. Lanes 5, 6, and 7 were urine from CPS-injected mice collected at 30 min, 2 hours, and 8 hours post-injection, respectively. The volume of sample loaded into each lane was adjusted to contain an equal amount of CPS, approximately 1 μg/lane. After blotting, membranes were probed with mAb 4C4 (1 μg/mL). Intact CPS was observed in urine samples from CPS-treated mice.

Techniques Used: Western Blot, Purification, SDS Page, Incubation, Injection, Mouse Assay

4) Product Images from "The Spike Protein of Murine Coronavirus Regulates Viral Genome Transport from the Cell Surface to the Endoplasmic Reticulum during Infection "

Article Title: The Spike Protein of Murine Coronavirus Regulates Viral Genome Transport from the Cell Surface to the Endoplasmic Reticulum during Infection

Journal: Journal of Virology

doi: 10.1128/JVI.00956-09

Parental A59 and recombinant Penn-98-1 viruses enter cells at similar levels. (A) Virus internalization assay. DBT cells were infected with radiolabeled MHV strains A59 and Penn-98-1, with equivalent levels of radioactivity. Viral genomic RNAs were prelabeled with [ 3 H]uridine, and the radiolabeled viruses were purified as described in Materials and Methods. Virus attachment was carried out at 4°C for 1 h. Following extensive washing of unbound viruses, cell cultures were either kept at 4°C as a negative control or moved to 37°C for an additional 45 min, the latter of which allows the virus to internalize. Bound but uninternalized viruses on the cell surface were then removed by treatment with protease K. Cells were then lysed, and intracellular radioactivity was determined with a liquid scintillation counter. The results are expressed as the mean counts per minute from three independent experiments. Error bars indicate the standard deviations of the means. (B) Infectious-center assay. Equivalent numbers of DBT cells were infected with MHV A59 and Penn-98-1 at an MOI of 5 at 4°C for 1 h to synchronize the infections. Cells were then moved to 37°C for 1 h to allow virus to enter the cells. Infected cells that remained at 4°C were used as a negative control. An infectious-center assay was then performed to determine the number of infected cells (infectious centers) for the original virus inoculum, as described in Materials and Methods. Error bars indicate the standard deviations of the means.
Figure Legend Snippet: Parental A59 and recombinant Penn-98-1 viruses enter cells at similar levels. (A) Virus internalization assay. DBT cells were infected with radiolabeled MHV strains A59 and Penn-98-1, with equivalent levels of radioactivity. Viral genomic RNAs were prelabeled with [ 3 H]uridine, and the radiolabeled viruses were purified as described in Materials and Methods. Virus attachment was carried out at 4°C for 1 h. Following extensive washing of unbound viruses, cell cultures were either kept at 4°C as a negative control or moved to 37°C for an additional 45 min, the latter of which allows the virus to internalize. Bound but uninternalized viruses on the cell surface were then removed by treatment with protease K. Cells were then lysed, and intracellular radioactivity was determined with a liquid scintillation counter. The results are expressed as the mean counts per minute from three independent experiments. Error bars indicate the standard deviations of the means. (B) Infectious-center assay. Equivalent numbers of DBT cells were infected with MHV A59 and Penn-98-1 at an MOI of 5 at 4°C for 1 h to synchronize the infections. Cells were then moved to 37°C for 1 h to allow virus to enter the cells. Infected cells that remained at 4°C were used as a negative control. An infectious-center assay was then performed to determine the number of infected cells (infectious centers) for the original virus inoculum, as described in Materials and Methods. Error bars indicate the standard deviations of the means.

Techniques Used: Recombinant, Infection, Radioactivity, Purification, Negative Control

5) Product Images from "Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis"

Article Title: Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis

Journal: Veterinary Sciences

doi: 10.3390/vetsci6040088

Preparation of EtOH extract for mass spectrometry analysis. ( a ) Map bacilli were washed 6 times prior to EtOH extraction (labeled × 6) to avoid bovine serum albumin (BSA) contamination. A second extract was prepared after washing Map once (label × 1). Lipids and carbohydrate were removed from the EtOH extract through a second chloroform:methanol:water extraction. This second extraction was run on 12% SDS-PAGE denaturing gels and exposed to Coomassie stain (CBB) and silver stain. The location of BSA migration is identified for both gel images. ( b ) Proteinase K treatment of Map EtOH extract. Inclusion of proteinase K or the EtOH extract along with staining method is indicated beneath the gel. Kilodalton size markers are shown in the left margins of all gels.
Figure Legend Snippet: Preparation of EtOH extract for mass spectrometry analysis. ( a ) Map bacilli were washed 6 times prior to EtOH extraction (labeled × 6) to avoid bovine serum albumin (BSA) contamination. A second extract was prepared after washing Map once (label × 1). Lipids and carbohydrate were removed from the EtOH extract through a second chloroform:methanol:water extraction. This second extraction was run on 12% SDS-PAGE denaturing gels and exposed to Coomassie stain (CBB) and silver stain. The location of BSA migration is identified for both gel images. ( b ) Proteinase K treatment of Map EtOH extract. Inclusion of proteinase K or the EtOH extract along with staining method is indicated beneath the gel. Kilodalton size markers are shown in the left margins of all gels.

Techniques Used: Mass Spectrometry, Labeling, SDS Page, Staining, Silver Staining, Migration

Bovine antibody binds to a carbohydrate component of the Map EtOH extract, not to protein. ( a ) Surface antigens of Map K-10 were extracted with 80% ethanol (EtOH Whole) and fractionated by Folch extraction method into organic (chloroform), interface and aqueous fractions. After evaporating methanol for immobilization of the lipids (and other molecules) onto the wells of a microtitre plate, they were incubated with serum samples (1:100 dilution) collected from JD-positive (solid bar) and negative (open bar) cattle. Histogram bars represent mean antibody binding ± standard deviation of quadruplicate determinations. Most of the antigenicity is in the organic fraction. ( b ) EtOH extract treated with proteases shows no negative effects on bovine antibody binding. The extract was treated with either 10 µg/mL trypsin or 10 µg/mL proteinase K with each treatment actually enhancing antibody binding. This enhancement was not statistically significant. However, EtOH extract antigens are efficiently removed by ConA-agarose as shown by lack of antibody binding ( c ). Absorption with agarose only does not affect antibody binding to the EtOH extract. This experiment was repeated in triplicate with qraduplicate measurements for each. p values less than 0.01 are denoted by an asterisk.
Figure Legend Snippet: Bovine antibody binds to a carbohydrate component of the Map EtOH extract, not to protein. ( a ) Surface antigens of Map K-10 were extracted with 80% ethanol (EtOH Whole) and fractionated by Folch extraction method into organic (chloroform), interface and aqueous fractions. After evaporating methanol for immobilization of the lipids (and other molecules) onto the wells of a microtitre plate, they were incubated with serum samples (1:100 dilution) collected from JD-positive (solid bar) and negative (open bar) cattle. Histogram bars represent mean antibody binding ± standard deviation of quadruplicate determinations. Most of the antigenicity is in the organic fraction. ( b ) EtOH extract treated with proteases shows no negative effects on bovine antibody binding. The extract was treated with either 10 µg/mL trypsin or 10 µg/mL proteinase K with each treatment actually enhancing antibody binding. This enhancement was not statistically significant. However, EtOH extract antigens are efficiently removed by ConA-agarose as shown by lack of antibody binding ( c ). Absorption with agarose only does not affect antibody binding to the EtOH extract. This experiment was repeated in triplicate with qraduplicate measurements for each. p values less than 0.01 are denoted by an asterisk.

Techniques Used: Incubation, Binding Assay, Standard Deviation

Related Articles

Incubation:

Article Title: RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs
Article Snippet: .. Each sample was then incubated for 60 min at 50 °C in 200 μL PK/SDS buffer (100 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.2% SDS) supplemented with 10 μL proteinase K (Fisher Scientific YSJ-762-Q). .. RNA-protein complexes were recovered by Phenol:Chloroform extraction and ethanol precipitation.

Article Title: Maelstrom Represses Canonical Polymerase II Transcription within Bi-Directional piRNA Clusters in Drosophila melanogaster
Article Snippet: .. Beads were then treated with 20 μg/ml RNase A (Fisher Scientific, #FEREN0531) To reverse crosslink and remove protein, beads were incubated overnight at 65°C with 200 μg/ml Proteinase K (Life Technologies, #25530015) in 2×Proteinase K Buffer (200 mM Tris-HCl [pH 7.5], 2 mM EDTA [pH 8.0], and 1% SDS (w/v). .. Finally, DNA was purified using phenol:chloroform [pH 8.0] and the library was prepared by sequentially performing end-repair, A-tailing, Y-shaped adaptor ligation, and PCR amplification as described ( ).

Generated:

Article Title: Evidence That Bank Vole PrP Is a Universal Acceptor for Prions
Article Snippet: .. Proteinase K digestions Ten percent (wt/vol) brain homogenates in calcium- and magnesium-free PBS were generated using either an OmniTip (Omni International) with a PowerGen homogenizer (Fisher Scientific) or with a bead beater (Precellys). .. Nine volumes of 10% brain homogenate were added to one volume of 10× detergent buffer [5% (vol/vol) NP-40, 5% (wt/vol) sodium deoxycholate in PBS] and then incubated on ice for 20 min followed by centrifugation at 1,000 × g for 5 min to remove cellular debris.

Electrophoresis:

Article Title: Potent and rapid activation of tropomyosin-receptor kinase A in endometrial stromal fibroblasts by seminal plasma
Article Snippet: Boiled proteinase K–treated and control samples were then further diluted 1:400 into 1X Laemmli buffer, and equal volumes of the samples were loaded onto a 15% Tris-HCl gel (Bio-Rad). .. After electrophoresis, protein degradation was visualized using the Pierce Silver Stain kit (Fisher Scientific), following the manufacturer's protocol. ..

Silver Staining:

Article Title: Potent and rapid activation of tropomyosin-receptor kinase A in endometrial stromal fibroblasts by seminal plasma
Article Snippet: Boiled proteinase K–treated and control samples were then further diluted 1:400 into 1X Laemmli buffer, and equal volumes of the samples were loaded onto a 15% Tris-HCl gel (Bio-Rad). .. After electrophoresis, protein degradation was visualized using the Pierce Silver Stain kit (Fisher Scientific), following the manufacturer's protocol. ..

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    Fisher Scientific proteinase k
    ZnO NPs affect biofilm formation. A.  The pellicle column depicts microtiter wells (6-well plate) in which cells were grown in biofilm medium with various concentrations of ZnO NPs at 25°C for 3 days (scale bar: 2 cm). Bacterial wild-type (3610) and mutant strains are indicated as follows:  sinR  (DS92),  epsA-O  (DS696),  sfp  (DS3629),  tasA  (DS3630), and  sinR epsA-O  (HS222).  B.  Images of a 12-well microtiter dish containing ethanol-precipitated supernatant from the indicated strain, following treatment with different concentrations of ZnO NPs.  C . The supernatants of the indicated strains were treated with proteinase K, DNase, and RNase, precipitated with ethanol, and resolved through SDS-PAGE on a 12% gel, after which staining with Stains-All was performed.  D.  FT-IR spectra analysis of EPS from  Bacillus  cells treated with ZnO NPs. Wild type bacteria were grown at ZnO NP concentrations of 0, 5, 10, 25, and 50 ppm, and untreated  eps  mutant cells were grown to serve as a negative control.
    Proteinase K, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase k/product/Fisher Scientific
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase k - by Bioz Stars, 2021-03
    86/100 stars
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    ZnO NPs affect biofilm formation. A.  The pellicle column depicts microtiter wells (6-well plate) in which cells were grown in biofilm medium with various concentrations of ZnO NPs at 25°C for 3 days (scale bar: 2 cm). Bacterial wild-type (3610) and mutant strains are indicated as follows:  sinR  (DS92),  epsA-O  (DS696),  sfp  (DS3629),  tasA  (DS3630), and  sinR epsA-O  (HS222).  B.  Images of a 12-well microtiter dish containing ethanol-precipitated supernatant from the indicated strain, following treatment with different concentrations of ZnO NPs.  C . The supernatants of the indicated strains were treated with proteinase K, DNase, and RNase, precipitated with ethanol, and resolved through SDS-PAGE on a 12% gel, after which staining with Stains-All was performed.  D.  FT-IR spectra analysis of EPS from  Bacillus  cells treated with ZnO NPs. Wild type bacteria were grown at ZnO NP concentrations of 0, 5, 10, 25, and 50 ppm, and untreated  eps  mutant cells were grown to serve as a negative control.

    Journal: PLoS ONE

    Article Title: ZnO Nanoparticles Affect Bacillus subtilis Cell Growth and Biofilm Formation

    doi: 10.1371/journal.pone.0128457

    Figure Lengend Snippet: ZnO NPs affect biofilm formation. A. The pellicle column depicts microtiter wells (6-well plate) in which cells were grown in biofilm medium with various concentrations of ZnO NPs at 25°C for 3 days (scale bar: 2 cm). Bacterial wild-type (3610) and mutant strains are indicated as follows: sinR (DS92), epsA-O (DS696), sfp (DS3629), tasA (DS3630), and sinR epsA-O (HS222). B. Images of a 12-well microtiter dish containing ethanol-precipitated supernatant from the indicated strain, following treatment with different concentrations of ZnO NPs. C . The supernatants of the indicated strains were treated with proteinase K, DNase, and RNase, precipitated with ethanol, and resolved through SDS-PAGE on a 12% gel, after which staining with Stains-All was performed. D. FT-IR spectra analysis of EPS from Bacillus cells treated with ZnO NPs. Wild type bacteria were grown at ZnO NP concentrations of 0, 5, 10, 25, and 50 ppm, and untreated eps mutant cells were grown to serve as a negative control.

    Article Snippet: Briefly, 1 ml of supernatant from overnight culture was treated with DNase and RNase to a final concentration of 67 μg/ml DNase I (Roche Diagnostics, Indianapolis, IN, USA) and 330 μg/ml Ribonuclease A (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 37°C, and subsequently treated with proteinase K to a final concentration of 400 μg/ml (Fisher Scientific, Rockford, IL, USA) for 1 h at 55°C.

    Techniques: Mutagenesis, SDS Page, Staining, Negative Control

    Confirmation of surface localization of DbpA in B. burgdorferi expressing lysine point mutants. BbKH500, BbKH501, BbDbpA K82A , BbDbpA K163A , and BbDbpA K170A were grown in vitro at pH 7.5 to the late exponential growth phase. Whole-cell lysates from intact bacteria subjected to proteinase K digestion (+) or mock treated (−) were analyzed by immunoblotting. Antibodies used to detect the respective proteins are indicated on the right. The values on the left denote relevant molecular masses (kDa) of the standard (lane M). Samples from three independent biological replicates were analyzed and shown to yield similar results. Depicted is an immunoblot from one of these biological replicates.

    Journal: Infection and Immunity

    Article Title: Identification of Lysine Residues in the Borrelia burgdorferi DbpA Adhesin Required for Murine Infection

    doi: 10.1128/IAI.02036-14

    Figure Lengend Snippet: Confirmation of surface localization of DbpA in B. burgdorferi expressing lysine point mutants. BbKH500, BbKH501, BbDbpA K82A , BbDbpA K163A , and BbDbpA K170A were grown in vitro at pH 7.5 to the late exponential growth phase. Whole-cell lysates from intact bacteria subjected to proteinase K digestion (+) or mock treated (−) were analyzed by immunoblotting. Antibodies used to detect the respective proteins are indicated on the right. The values on the left denote relevant molecular masses (kDa) of the standard (lane M). Samples from three independent biological replicates were analyzed and shown to yield similar results. Depicted is an immunoblot from one of these biological replicates.

    Article Snippet: Spirochetes (1 × 109 ) were treated with 200 μg of proteinase K (10 mg/ml; Fisher Scientific, Pittsburgh, PA) or sham treated for 60 min at room temperature.

    Techniques: Expressing, In Vitro

    Negative-strand product RNA is linked to VPg. (A) PIRCs formed with PV1 RNA were resuspended in reaction mixtures containing [α- 32 P]CTP and were incubated for 1 h at 37°C. The 32 P-labeled product RNA was phenol extracted and ethanol precipitated. Half of the product RNA was treated with proteinase K to remove VPg as described in Materials and Methods. The labeled product RNAs were then immunoprecipitated with anti-VPg antibody (Ab) and analyzed by CH 3 HgOH-agarose gel electrophoresis. (B) PIRCs formed with PV1 cre (2C)mut1 RNA or PV1 RNA were resuspended in reaction reactions containing [α- 32 P]CTP and incubated at 37°C for 1 h. The 32 P-labeled product RNAs were phenol extracted, ethanol precipitated, and immunoprecipitated with anti-VPg antibody. The immunoprecipitated products were analyzed by CH 3 HgOH-agarose gel electrophoresis.

    Journal: Journal of Virology

    Article Title: Poliovirus cre(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis

    doi: 10.1128/JVI.77.9.5136-5144.2003

    Figure Lengend Snippet: Negative-strand product RNA is linked to VPg. (A) PIRCs formed with PV1 RNA were resuspended in reaction mixtures containing [α- 32 P]CTP and were incubated for 1 h at 37°C. The 32 P-labeled product RNA was phenol extracted and ethanol precipitated. Half of the product RNA was treated with proteinase K to remove VPg as described in Materials and Methods. The labeled product RNAs were then immunoprecipitated with anti-VPg antibody (Ab) and analyzed by CH 3 HgOH-agarose gel electrophoresis. (B) PIRCs formed with PV1 cre (2C)mut1 RNA or PV1 RNA were resuspended in reaction reactions containing [α- 32 P]CTP and incubated at 37°C for 1 h. The 32 P-labeled product RNAs were phenol extracted, ethanol precipitated, and immunoprecipitated with anti-VPg antibody. The immunoprecipitated products were analyzed by CH 3 HgOH-agarose gel electrophoresis.

    Article Snippet: In the proteinase K-treated control, the phenol-extracted labeled product RNA was resuspended in 200 μl of 0.5% SDS buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 0.5% SDS) and incubated at 37°C for 45 min with 1 μl of 20 mg of proteinase K per ml (Fisher Scientific) to digest covalently linked VPg.

    Techniques: Incubation, Labeling, Immunoprecipitation, Agarose Gel Electrophoresis

    NAMPT and NMNAT3 are soluble matrix proteins. (a-b) Mitochondria isolated from mouse primary cultured cortical neurons (a) and mouse cortex (b) were digested by proteinase K with different concentrations for 40 min on ice and then immunoblotted for Mfn1, OPA1, AtpS C, C1qbp, NAMPT and NMANT3. OM-outer membrane, InterM-Inter membrane space, IM-Inner membrane, MM-mitochondrial matrix. Data are representative images from n=3 different mitochondrial subfractionation of neuronal culture and n=4 mouse cortical tissue. (c) Schematic illustration of mitochondrial NAD + salvage pathway in neurons. Based on our results, both NAMPT and NMNAT3 are expressed in the mitochondria and are localized in the mitochondrial matrix. We propose neuronal mitochondria have an intact NAD + salvage pathway and the substrates NAM and PRPP can be transported from cytoplasm or synthesized within the organelle (route 1). For mammalian cells lacking NAMPT expression in the mitochondria, NMN is transported into mitochondria for NAD + ).

    Journal: Journal of neurochemistry

    Article Title: Subcellular NAMPT-mediated NAD+ salvage pathways and their roles in bioenergetics and neuronal protection after ischemic injury

    doi: 10.1111/jnc.14878

    Figure Lengend Snippet: NAMPT and NMNAT3 are soluble matrix proteins. (a-b) Mitochondria isolated from mouse primary cultured cortical neurons (a) and mouse cortex (b) were digested by proteinase K with different concentrations for 40 min on ice and then immunoblotted for Mfn1, OPA1, AtpS C, C1qbp, NAMPT and NMANT3. OM-outer membrane, InterM-Inter membrane space, IM-Inner membrane, MM-mitochondrial matrix. Data are representative images from n=3 different mitochondrial subfractionation of neuronal culture and n=4 mouse cortical tissue. (c) Schematic illustration of mitochondrial NAD + salvage pathway in neurons. Based on our results, both NAMPT and NMNAT3 are expressed in the mitochondria and are localized in the mitochondrial matrix. We propose neuronal mitochondria have an intact NAD + salvage pathway and the substrates NAM and PRPP can be transported from cytoplasm or synthesized within the organelle (route 1). For mammalian cells lacking NAMPT expression in the mitochondria, NMN is transported into mitochondria for NAD + ).

    Article Snippet: To assess sub-mitochondrial compartment localization of NAMPT, the purified mitochondrial pellet was digested by proteinase K (0.1–25.6 μg/ml) (Cat. No. , Thermal Fisher Scientific) in PBS for 45 min on ice ( ).

    Techniques: Isolation, Cell Culture, Synthesized, Expressing