tris edta buffer  (Millipore)

 
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    Name:
    Trizma base
    Description:
    Tris is an established basimetric standard and buffer used in biochemistry and molecular biology It may be used by itself as a buffer or as a component of mixed buffer formulations such as Tris EDTA TE buffer Tris acetate EDTA TAE buffer Tris borate EDTA TBE buffer etc It is pure essentially stable relatively non hygroscopic and has a high equivalent weight
    Catalog Number:
    T4661
    Price:
    None
    Applications:
    Tris base has been used:. As a component of lysis buffer for cell disruption. In the preparation of TBE solution for PAGE (polyacrylamide gel electrophoresis). In studies of double stranded complexes of peptide nucleic acids (PNA) and their complementary DNA sequences, by use of anion exchange HPLC. In capillary electrochromatography and UV analysis of tocopherols and tocotrienols
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    Structured Review

    Millipore tris edta buffer
    Trizma base
    Tris is an established basimetric standard and buffer used in biochemistry and molecular biology It may be used by itself as a buffer or as a component of mixed buffer formulations such as Tris EDTA TE buffer Tris acetate EDTA TAE buffer Tris borate EDTA TBE buffer etc It is pure essentially stable relatively non hygroscopic and has a high equivalent weight
    https://www.bioz.com/result/tris edta buffer/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tris edta buffer - by Bioz Stars, 2021-04
    99/100 stars

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    other:

    Article Title: A Colorimetric Assay Method to Measure Acetyl-CoA Synthetase Activity: Application to Woodchuck Model of Hepatitis Virus-induced Hepatocellular Carcinoma
    Article Snippet: Acetyl-CoA synthetase, CoA, dithiothreitol (DTT), ATP, Trizma base, ammonium molydate, sodium pyrophosphate, sodium phosphate dibasic, thioglycolic acid, 2-mercaptoethanol and 1-amino-2-naphthol-4-sulfonic acid were purchased from Sigma Chemical Co. (St. Louis, MO).

    Article Title: Characterization of the putative chloride channel xClC-5 expressed in Xenopus laevis oocytes and comparison with endogenous chloride currents
    Article Snippet: 2( N -Morpholino)ethanesulphonic acid) (Mes) and Trizma base, used in the investigation of pH effects, were from Sigma.

    Alkaline Lysis:

    Article Title: K13-Mediated Reduced Susceptibility to Artemisinin in Plasmodium falciparum Is Overlaid on a Trait of Enhanced DNA Damage Repair
    Article Snippet: Alkaline lysis for the CometChip was performed at 4°C overnight. .. Alkaline lysis buffer (pH ~10) consisted of 2.5 M Sodium chloride, 100 mM disodium EDTA, 10 mM Trizma® base, and 1% v/v Triton X-100 (Sigma-Aldrich, USA) in deionized H2O. .. After alkaline lysis, the chip was submerged in cold alkaline electrophoresis buffer (pH ~13.5), containing 0.3 M sodium hydroxide and 1 mM disodium EDTA, to unwind the nuclei at 4°C for 40min.

    Polymerase Chain Reaction:

    Article Title: Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing
    Article Snippet: BD FACSFlow™ (Becton Dickinson GmbH, Cat. No. 322003) Flow-Check ™ Fluorospheres (Beckman Coulter, Cat No. 6605359) .. HotStar HighFidelity PCR kit (QIAGEN, cat. no. 202605) Trizma® base (Sigma, cat.no. .. T1503) TWEEN® 20 (Merck, cat. P1379) Triton X100 (Merck, cat. T9284) MgCl2 (Merck, cat. M8266) Proteinase K (Merck, cat. P2308 )

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing
    Article Snippet: BD FACSFlow™ (Becton Dickinson GmbH, Cat. No. 322003) Flow-Check ™ Fluorospheres (Beckman Coulter, Cat No. 6605359) .. HotStar HighFidelity PCR kit (QIAGEN, cat. no. 202605) Trizma® base (Sigma, cat.no. .. T1503) TWEEN® 20 (Merck, cat. P1379) Triton X100 (Merck, cat. T9284) MgCl2 (Merck, cat. M8266) Proteinase K (Merck, cat. P2308 )

    Flow Cytometry:

    Article Title: Isolation of a protein Z-dependent plasma protease inhibitor
    Article Snippet: The results of recent experiments reexamining this apparent PZ-mediated inhibition of factor Xa procoagulant activity show that PZ affects factor Xa function in part by enhancing the inhibition of factor Xa produced by another plasma protein we tentatively have termed the PZ-dependent protease inhibitor (ZPI). .. SDS, Hepes, Mes, Trizma base, diisopropyl fluorophosphate (DFP), Triton X-100, Tween 20, EDTA, polyethylene glycol ( M r , 8,000), S Fast Flow, BSA, and rabbit brain cephalin were from Sigma. ..

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    Millipore co immunoprecipitation buffer
    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 <t>co-immunoprecipitation</t> assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.
    Co Immunoprecipitation Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/co immunoprecipitation buffer/product/Millipore
    Average 97 stars, based on 1 article reviews
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    99
    Millipore tris edta
    ( a ) Concentration-dependent SERS spectra using AgNPs and <t>Tris-EDTA</t> in the range of 0.005–10 μM of Cr(III); ( b ) A magnified view of the vibrational bands between 200 and 750 cm −1 in the Cr(III) concentration-dependent SERS spectra; ( c ) A calibration curve of the vibrational band intensities at ~563 cm −1 with respect to those at ~918 cm −1 . The inset shows a linear fitting result for the 0.05 and 1.0 μM regions.
    Tris Edta, supplied by Millipore, 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/tris edta/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tris edta - by Bioz Stars, 2021-04
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    99
    Millipore buffer f
    Effect of loss of Rpn10/p54 (A and C) proteasome subunit or Usp5 deubiquitylase (B and C) on the abundance of ubiquitin forms. Whole protein extracts in <t>buffer</t> F (lanes 1, 2, 5 and 6; +NEM) and buffer T (lanes 3, 4, 7 and 8; -NEM) of wandering L3 larvae were investigated by western blotting using polyclonal anti-Ub antibody at a dilution of 1:1000. The bands just below the 10 kDa mark on the immunoblots (A and B) represent free monoubiquitins, and only the intensity of these bands were determined and used for quantification. 5 μg total protein extracts were loaded to lanes 2 and 6, while samples were diluted 1.7-fold to lanes 1 and 5; twofold to lanes 3, 4 and 8; and 3.3-fold to lane 7 before loading to avoid overloading the monoubiquitin band. Ubiquitin content (small table in C) was calculated by plotting band intensities against Ub standards and a regression line equation was generated by applying the four parameter curve fit model (R 2 = 0.9979 for Rpn10/p54 and R 2 = 0.9933 for Usp5), values were normalized to total protein content and shown as a column diagram.
    Buffer F, supplied by Millipore, 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/buffer f/product/Millipore
    Average 99 stars, based on 1 article reviews
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    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Binding Assay, Co-Immunoprecipitation Assay, SDS Page, Immunoprecipitation, Western Blot, Functional Assay

    AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Chemotaxis Assay, Sequencing, Functional Assay, Binding Assay, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

    Formation of C5aR homodimers as detected by co-immunoprecipitation analysis. C5aR and V5-tagged C5aR were co-expressed in HEK293 cells. Cells lysis was performed in nondenaturing detergent buffer. Receptors were immunoprecipitated ( IP ) with antibodies

    Journal:

    Article Title: Complement Component 5a Receptor Oligomerization and Homologous Receptor Down-regulation *

    doi: 10.1074/jbc.M805260200

    Figure Lengend Snippet: Formation of C5aR homodimers as detected by co-immunoprecipitation analysis. C5aR and V5-tagged C5aR were co-expressed in HEK293 cells. Cells lysis was performed in nondenaturing detergent buffer. Receptors were immunoprecipitated ( IP ) with antibodies

    Article Snippet: Two days after transfection, cells were lysed in nondenaturing ice-cold buffer (50 m m Tris, pH 8.0, 150 m m NaCl, 1 m m EDTA, 1% Nonidet P-40, 15% glycerol) containing protease and phosphatase inhibitors (protease inhibitor mixture and phosphatase inhibitor mixture; Sigma).

    Techniques: Immunoprecipitation, Lysis

    ( a ) Concentration-dependent SERS spectra using AgNPs and Tris-EDTA in the range of 0.005–10 μM of Cr(III); ( b ) A magnified view of the vibrational bands between 200 and 750 cm −1 in the Cr(III) concentration-dependent SERS spectra; ( c ) A calibration curve of the vibrational band intensities at ~563 cm −1 with respect to those at ~918 cm −1 . The inset shows a linear fitting result for the 0.05 and 1.0 μM regions.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Silver Nanoparticle-Enhanced Resonance Raman Sensor of Chromium(III) in Seawater Samples

    doi: 10.3390/s150510088

    Figure Lengend Snippet: ( a ) Concentration-dependent SERS spectra using AgNPs and Tris-EDTA in the range of 0.005–10 μM of Cr(III); ( b ) A magnified view of the vibrational bands between 200 and 750 cm −1 in the Cr(III) concentration-dependent SERS spectra; ( c ) A calibration curve of the vibrational band intensities at ~563 cm −1 with respect to those at ~918 cm −1 . The inset shows a linear fitting result for the 0.05 and 1.0 μM regions.

    Article Snippet: Chemicals Cr(III) and the other ionic substances of NaCl, KNO3 , Mg(NO3 )2 , Ca(NO3 )2 , Cr(NO3 )3 , Mn(NO3 )2 , FeC2 O4 , Fe(NO3 )3 , Co(NO3 )2 , Ni(NO3 )2 , Cu(NO3 )2 , Zn(NO3 )2 , NH4 NO3 , Cd(NO3 )2 , Hg(NO3 )2 , Pb(NO3 )2 , and K2 Cr2 O7 (or Na2 CrO4 ) along with silver nitrate, sodium citrate, Tris-EDTA (pH 8.0), and EDTA acetic acid were purchased from Sigma Aldrich (St. Louis, MO, USA).

    Techniques: Concentration Assay

    ( a ) Surface-enhanced Raman spectroscopy (SERS) spectra of detection of Cr(III) using Tris(hydroxymethyl)aminomethane (Tris)-EDTA on AgNPs. Selective tests were performed for 50 μM of Cr 3+ , K + , Cd 2+ , Mg 2+ , Ca 2+ , Mn 2+ , Co 2+ , Na + , Cu 2+ , NH 4 + , Hg 2+ , Ni 2+ , Fe 3+ , Pb 2+ , Fe 2+ , and Zn 2+ ions; ( b ) Stick diagram of the intensities at ~563 cm −1 with respect to those at ~918 cm −1 versus 16 ionic species; ( c ) SERS spectra of the mixture of the 15 ions to test the competitive reactions; ( d ) Comparative SERS spectra of EDTA and EDTA-Cr 3+ on AgNPs at pH 4.0 and pH 8.0.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Silver Nanoparticle-Enhanced Resonance Raman Sensor of Chromium(III) in Seawater Samples

    doi: 10.3390/s150510088

    Figure Lengend Snippet: ( a ) Surface-enhanced Raman spectroscopy (SERS) spectra of detection of Cr(III) using Tris(hydroxymethyl)aminomethane (Tris)-EDTA on AgNPs. Selective tests were performed for 50 μM of Cr 3+ , K + , Cd 2+ , Mg 2+ , Ca 2+ , Mn 2+ , Co 2+ , Na + , Cu 2+ , NH 4 + , Hg 2+ , Ni 2+ , Fe 3+ , Pb 2+ , Fe 2+ , and Zn 2+ ions; ( b ) Stick diagram of the intensities at ~563 cm −1 with respect to those at ~918 cm −1 versus 16 ionic species; ( c ) SERS spectra of the mixture of the 15 ions to test the competitive reactions; ( d ) Comparative SERS spectra of EDTA and EDTA-Cr 3+ on AgNPs at pH 4.0 and pH 8.0.

    Article Snippet: Chemicals Cr(III) and the other ionic substances of NaCl, KNO3 , Mg(NO3 )2 , Ca(NO3 )2 , Cr(NO3 )3 , Mn(NO3 )2 , FeC2 O4 , Fe(NO3 )3 , Co(NO3 )2 , Ni(NO3 )2 , Cu(NO3 )2 , Zn(NO3 )2 , NH4 NO3 , Cd(NO3 )2 , Hg(NO3 )2 , Pb(NO3 )2 , and K2 Cr2 O7 (or Na2 CrO4 ) along with silver nitrate, sodium citrate, Tris-EDTA (pH 8.0), and EDTA acetic acid were purchased from Sigma Aldrich (St. Louis, MO, USA).

    Techniques: Raman Spectroscopy

    Effect of loss of Rpn10/p54 (A and C) proteasome subunit or Usp5 deubiquitylase (B and C) on the abundance of ubiquitin forms. Whole protein extracts in buffer F (lanes 1, 2, 5 and 6; +NEM) and buffer T (lanes 3, 4, 7 and 8; -NEM) of wandering L3 larvae were investigated by western blotting using polyclonal anti-Ub antibody at a dilution of 1:1000. The bands just below the 10 kDa mark on the immunoblots (A and B) represent free monoubiquitins, and only the intensity of these bands were determined and used for quantification. 5 μg total protein extracts were loaded to lanes 2 and 6, while samples were diluted 1.7-fold to lanes 1 and 5; twofold to lanes 3, 4 and 8; and 3.3-fold to lane 7 before loading to avoid overloading the monoubiquitin band. Ubiquitin content (small table in C) was calculated by plotting band intensities against Ub standards and a regression line equation was generated by applying the four parameter curve fit model (R 2 = 0.9979 for Rpn10/p54 and R 2 = 0.9933 for Usp5), values were normalized to total protein content and shown as a column diagram.

    Journal: PLoS ONE

    Article Title: Developmental and tissue specific changes of ubiquitin forms in Drosophila melanogaster

    doi: 10.1371/journal.pone.0209080

    Figure Lengend Snippet: Effect of loss of Rpn10/p54 (A and C) proteasome subunit or Usp5 deubiquitylase (B and C) on the abundance of ubiquitin forms. Whole protein extracts in buffer F (lanes 1, 2, 5 and 6; +NEM) and buffer T (lanes 3, 4, 7 and 8; -NEM) of wandering L3 larvae were investigated by western blotting using polyclonal anti-Ub antibody at a dilution of 1:1000. The bands just below the 10 kDa mark on the immunoblots (A and B) represent free monoubiquitins, and only the intensity of these bands were determined and used for quantification. 5 μg total protein extracts were loaded to lanes 2 and 6, while samples were diluted 1.7-fold to lanes 1 and 5; twofold to lanes 3, 4 and 8; and 3.3-fold to lane 7 before loading to avoid overloading the monoubiquitin band. Ubiquitin content (small table in C) was calculated by plotting band intensities against Ub standards and a regression line equation was generated by applying the four parameter curve fit model (R 2 = 0.9979 for Rpn10/p54 and R 2 = 0.9933 for Usp5), values were normalized to total protein content and shown as a column diagram.

    Article Snippet: The animal and tissue samples were homogenized by plastic tissue grinders in pre-chilled microfuge tubes either in 100 μl ice cold buffer F [100 mM Tris, pH 7.6, 150 mM NaCl, 1mM EDTA, 10 mM N-Ethylmaleimide (NEM, Sigma-Aldrich), 20μM MG132 (Calbiochem) and 1× EDTA-Free Complete Protease Inhibitor Cocktail (Roche)] or 100 μl buffer T [Buffer F supplemented with 2mM DTT to preserve the catalytic activity of DUBs, but without NEM], respectively.

    Techniques: Western Blot, Generated