Structured Review

GE Healthcare leupeptin
Cidea is a short-lived protein and its degradation is proteasome-dependent ( A , B and C ) A 1 μg portion of CMV5–HA–hCidea was co-transfected with 0.5 μg of pEGFP-N1 into HEK-293T, CHO-K1 and H1299 cells in 60-mm-diameter dishes using Dosper reagent. CHX-based protein chase experiments were performed as described below. At 24 h post-transfection, and 1 h prior to the addition of CHX, the medium was replaced with fresh medium, and then CHX was added to a final concentration of 100 μg/ml. Cells were harvested in 0.5 ml of lysis buffer at different time points: 0, 30, 60 and 120 min. Total cell lysates were prepared and analysed by Western blotting [IB (immunoblotting)] using anti-HA and anti-GFP as primary antibodies. The amount of GFP transiently co-expressed by pEGFP-N1 present in total cell lysates was used for the normalization of transfection protein. ( D ) Graph showing band intensities corresponding to HA–Cidea and GFP scanned and quantitified using ImageQuant-TL software (Amersham). Results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The ratio was set to 100% at zero time. The results shown are representative of three independent experiments. ( E ) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1 μg of CMV-HA vector (lane 1) or 1 μg of CMV5–HA–hCidea (lane 2–11) and 0.5 μg of pEGFP-N1 using the calcium phosphate method. After 24 h, CHX was added to 100 μg/ml (lanes 3–11) along with different reagents [DMSO, ethanol, pepstatin (0.5 μg/ml), <t>leupeptin</t> (5 μg/ml), aprotinin (2 μg/ml), ALLN (50 μM), MG132 (10 μM), chloroquine (50 μg/ml) and NH 4 Cl (2.5 mM)] for 2 h. Cells were harvested in 0.5 ml of lysis buffer after the 2 h treatment. Total cell lysates were subjected to Western blotting and detected with anti-HA and anti-GFP antibodies.
Leupeptin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/leupeptin/product/GE Healthcare
Average 92 stars, based on 29 article reviews
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leupeptin - by Bioz Stars, 2020-07
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1) Product Images from "Regulation of Cidea protein stability by the ubiquitin-mediated proteasomal degradation pathway"

Article Title: Regulation of Cidea protein stability by the ubiquitin-mediated proteasomal degradation pathway

Journal: The Biochemical Journal

doi: 10.1042/BJ20070690

Cidea is a short-lived protein and its degradation is proteasome-dependent ( A , B and C ) A 1 μg portion of CMV5–HA–hCidea was co-transfected with 0.5 μg of pEGFP-N1 into HEK-293T, CHO-K1 and H1299 cells in 60-mm-diameter dishes using Dosper reagent. CHX-based protein chase experiments were performed as described below. At 24 h post-transfection, and 1 h prior to the addition of CHX, the medium was replaced with fresh medium, and then CHX was added to a final concentration of 100 μg/ml. Cells were harvested in 0.5 ml of lysis buffer at different time points: 0, 30, 60 and 120 min. Total cell lysates were prepared and analysed by Western blotting [IB (immunoblotting)] using anti-HA and anti-GFP as primary antibodies. The amount of GFP transiently co-expressed by pEGFP-N1 present in total cell lysates was used for the normalization of transfection protein. ( D ) Graph showing band intensities corresponding to HA–Cidea and GFP scanned and quantitified using ImageQuant-TL software (Amersham). Results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The ratio was set to 100% at zero time. The results shown are representative of three independent experiments. ( E ) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1 μg of CMV-HA vector (lane 1) or 1 μg of CMV5–HA–hCidea (lane 2–11) and 0.5 μg of pEGFP-N1 using the calcium phosphate method. After 24 h, CHX was added to 100 μg/ml (lanes 3–11) along with different reagents [DMSO, ethanol, pepstatin (0.5 μg/ml), leupeptin (5 μg/ml), aprotinin (2 μg/ml), ALLN (50 μM), MG132 (10 μM), chloroquine (50 μg/ml) and NH 4 Cl (2.5 mM)] for 2 h. Cells were harvested in 0.5 ml of lysis buffer after the 2 h treatment. Total cell lysates were subjected to Western blotting and detected with anti-HA and anti-GFP antibodies.
Figure Legend Snippet: Cidea is a short-lived protein and its degradation is proteasome-dependent ( A , B and C ) A 1 μg portion of CMV5–HA–hCidea was co-transfected with 0.5 μg of pEGFP-N1 into HEK-293T, CHO-K1 and H1299 cells in 60-mm-diameter dishes using Dosper reagent. CHX-based protein chase experiments were performed as described below. At 24 h post-transfection, and 1 h prior to the addition of CHX, the medium was replaced with fresh medium, and then CHX was added to a final concentration of 100 μg/ml. Cells were harvested in 0.5 ml of lysis buffer at different time points: 0, 30, 60 and 120 min. Total cell lysates were prepared and analysed by Western blotting [IB (immunoblotting)] using anti-HA and anti-GFP as primary antibodies. The amount of GFP transiently co-expressed by pEGFP-N1 present in total cell lysates was used for the normalization of transfection protein. ( D ) Graph showing band intensities corresponding to HA–Cidea and GFP scanned and quantitified using ImageQuant-TL software (Amersham). Results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The ratio was set to 100% at zero time. The results shown are representative of three independent experiments. ( E ) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1 μg of CMV-HA vector (lane 1) or 1 μg of CMV5–HA–hCidea (lane 2–11) and 0.5 μg of pEGFP-N1 using the calcium phosphate method. After 24 h, CHX was added to 100 μg/ml (lanes 3–11) along with different reagents [DMSO, ethanol, pepstatin (0.5 μg/ml), leupeptin (5 μg/ml), aprotinin (2 μg/ml), ALLN (50 μM), MG132 (10 μM), chloroquine (50 μg/ml) and NH 4 Cl (2.5 mM)] for 2 h. Cells were harvested in 0.5 ml of lysis buffer after the 2 h treatment. Total cell lysates were subjected to Western blotting and detected with anti-HA and anti-GFP antibodies.

Techniques Used: Transfection, Concentration Assay, Lysis, Western Blot, Software, Plasmid Preparation

2) Product Images from "Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation"

Article Title: Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.314856

Recognition of VACV-infected cells by J6R(303–311)- or D1R(808–817)-specific CD8 + T cells in the presence of general protease inhibitors. Cells infected as described in were treated with leupeptin ( LEU ) (trypsin-like and cysteine
Figure Legend Snippet: Recognition of VACV-infected cells by J6R(303–311)- or D1R(808–817)-specific CD8 + T cells in the presence of general protease inhibitors. Cells infected as described in were treated with leupeptin ( LEU ) (trypsin-like and cysteine

Techniques Used: Infection

Related Articles

Centrifugation:

Article Title: A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs
Article Snippet: .. Cells were lysed with an IP buffer (50 mM Tris–HCl [pH 8.0], 250 mM NaCl, 0.5% Nonidet P-40, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF, 1 μg ml−1 aprotinin, 1 μg ml−1 leupeptin, and 1 μg ml−1 pepstatin) on ice for 30 min. After centrifugation, the supernatants were divided and incubated with the indicated antibodies for 1 h on ice, and then incubated with Protein G-Sepharose (17061801, GE Healthcare) for another 1 h at 4 °C. .. The immunoprecipitates were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (IPVH 00010, Millipore).

Purification:

Article Title: Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis
Article Snippet: .. Recombinant GST- or His-tagged proteins were expressed in E. coli (ArcticExpress RP, Agilent Technologies) with 0.5 mM IPTG at 12°C, solubilized in buffer A (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM 2-Mercaptoethanol, 1 mM PMSF and 1 µg/ml leupeptin) with 0.25 mg/ml lysozyme and 0.2 % NP-40, purified from the soluble fraction using glutathione-Sepharose 4B (GE Healthcare) or TALON Metal Affinity Resin (Clonetech), washed with buffer A containing 500 mM NaCl and 0.1% sodium cholate, and eluted with 20 mM glutathione or 500 mM imidazole, respectively. .. For His-tagged proteins, 5 mM imidazole was included in crude extracts to reduce nonspecific binding.

Incubation:

Article Title: A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs
Article Snippet: .. Cells were lysed with an IP buffer (50 mM Tris–HCl [pH 8.0], 250 mM NaCl, 0.5% Nonidet P-40, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF, 1 μg ml−1 aprotinin, 1 μg ml−1 leupeptin, and 1 μg ml−1 pepstatin) on ice for 30 min. After centrifugation, the supernatants were divided and incubated with the indicated antibodies for 1 h on ice, and then incubated with Protein G-Sepharose (17061801, GE Healthcare) for another 1 h at 4 °C. .. The immunoprecipitates were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (IPVH 00010, Millipore).

Article Title: The Non-Canonical CTD of RNAP-II Is Essential for Productive RNA Synthesis in Trypanosoma brucei
Article Snippet: .. For immunoprecipitations, nuclear extracts were incubated with anti-RPB4 antibody bound to protein A-sepharose, washed in buffer (20 mM Hepes [pH 7.9], 150 mM KCL, 150 sucrose, 2.5 mM MgCl2 , 1 mM EDTA, 2.5 mM DTT, 0.1%NP-40, 1 µM each of pepstatin A, leupeptin, and PMSF) and analyzed by immunoblotting using ECL™ kit from Amersham. .. RNA analysis Total cellular RNA from wild-type and transgenic cells was prepared using Trizol™ reagents.

Western Blot:

Article Title: Biosynthesis of Taxadiene in Saccharomyces cerevisiae : Selection of Geranylgeranyl Diphosphate Synthase Directed by a Computer-Aided Docking Strategy
Article Snippet: .. Western blotting Total proteins were prepared from yeast cells with an extraction buffer (2 mL/g cells) containing 50 mM 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid (HEPES) (pH 7.4), 10% glycerol (v/v), 10 mM EDTA, 0.1% Triton X-100 (v/v), 200 µM phenylmethyl sulfonyl fluoride (PMSF), and 2 µg/mL each of aprotinin, leupeptin, and pepstatin A. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels, and the polypeptides were transferred to PVDF membranes (0.22 µM, Amersham Life Science, Little Chalfon, UK) in a medium consisting of 25 mM Tris-HCl (pH 8.3), 192 mM glycine and 20% (v/v) methanol. .. After rinsing in the Tris-buffered saline (TBS) containing 10 mM Tris-HCl (pH 7.5) and 150 mM NaCl, the blotted membranes were pre-incubated for 2 h in a blocking buffer containing 5% (w/v) non-fat milk dissolved in TBS supplemented by 0.05% (v/v) Tween-20 (TBST1) and then incubated with gentle shaking for 2 h at room temperature in the appropriate antibodies (Anti-FLAG, Sigma, diluted 1∶10000 in the blocking buffer).

Affinity Purification:

Article Title: CAND1 regulates lunapark for the proper tubular network of the endoplasmic reticulum
Article Snippet: .. GST-Lnp, GST-Lnp Ub, GST-Rtn3c, and GST alone were expressed in E . coli BL21 (DE3) harbouring pGEX6P-1-Lnp, pGEX6P-1-Lnp Ub, pGEX6P-1-Rtn3c, and pGEX6P-1, respectively, and extracted with buffer A containing 1% Triton X-100, supplemented with 10 μM APMSF, 10 μg/ml leupeptin, and 5 μg/ml aprotinin, followed by affinity purification with glutathione Sepharose (GE Healthcare). ..

Recombinant:

Article Title: Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis
Article Snippet: .. Recombinant GST- or His-tagged proteins were expressed in E. coli (ArcticExpress RP, Agilent Technologies) with 0.5 mM IPTG at 12°C, solubilized in buffer A (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM 2-Mercaptoethanol, 1 mM PMSF and 1 µg/ml leupeptin) with 0.25 mg/ml lysozyme and 0.2 % NP-40, purified from the soluble fraction using glutathione-Sepharose 4B (GE Healthcare) or TALON Metal Affinity Resin (Clonetech), washed with buffer A containing 500 mM NaCl and 0.1% sodium cholate, and eluted with 20 mM glutathione or 500 mM imidazole, respectively. .. For His-tagged proteins, 5 mM imidazole was included in crude extracts to reduce nonspecific binding.

SDS Page:

Article Title: Biosynthesis of Taxadiene in Saccharomyces cerevisiae : Selection of Geranylgeranyl Diphosphate Synthase Directed by a Computer-Aided Docking Strategy
Article Snippet: .. Western blotting Total proteins were prepared from yeast cells with an extraction buffer (2 mL/g cells) containing 50 mM 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid (HEPES) (pH 7.4), 10% glycerol (v/v), 10 mM EDTA, 0.1% Triton X-100 (v/v), 200 µM phenylmethyl sulfonyl fluoride (PMSF), and 2 µg/mL each of aprotinin, leupeptin, and pepstatin A. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels, and the polypeptides were transferred to PVDF membranes (0.22 µM, Amersham Life Science, Little Chalfon, UK) in a medium consisting of 25 mM Tris-HCl (pH 8.3), 192 mM glycine and 20% (v/v) methanol. .. After rinsing in the Tris-buffered saline (TBS) containing 10 mM Tris-HCl (pH 7.5) and 150 mM NaCl, the blotted membranes were pre-incubated for 2 h in a blocking buffer containing 5% (w/v) non-fat milk dissolved in TBS supplemented by 0.05% (v/v) Tween-20 (TBST1) and then incubated with gentle shaking for 2 h at room temperature in the appropriate antibodies (Anti-FLAG, Sigma, diluted 1∶10000 in the blocking buffer).

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    GE Healthcare leupeptin
    Leupeptin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/leupeptin/product/GE Healthcare
    Average 92 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    leupeptin - by Bioz Stars, 2020-07
    92/100 stars
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