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4x laemmli sample buffer bio rad  (Bio-Rad)


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    Bio-Rad 4x laemmli sample buffer bio rad
    Targeted activation of endogenous mitochondrial ClpP. (A) Western blots of mitochondrial lysates (30 μg) from wild‐type and ClpP‐KO HEK293T cells were probed with antibodies against ClpP and α‐tubulin, the latter being used as a loading control. (B) Analysis of ClpP and ClpX assembly was carried out in wild‐type HEK293T mitochondria through isokinetic sucrose gradient centrifugation. Mitochondrial lysate (0.65 mg) was centrifuged on a 10%–30% gradient using the SW 60Ti rotor at 40 000 rpm for 2 h and 15 min at 4°C. Small and large mitoribosomal subunits were used as a reference of migration and were immunodecorated by antibodies against marker proteins of the mt‐SSU (mS29; HUGO protein name: DAP3) and the mt‐LSU (uL13m; HUGO protein name: MRPL13). (C) Effect of ONC201‐mediated ClpP activation on casein hydrolysis in the PAGE‐based fluorescent assay. Mitochondrial lysates (50 μg) from wild‐type and KO cells were used as a source of ClpP activity, and incubated with ONC201 at a final concentration of 100 μM. Upper panels: Milk casein isoforms (α, β and k) are depicted to the left; molecular weight markers (M; Precision Plus Protein Dual Color Standards, <t>Bio‐Rad)</t> are indicated to the right. Middle panels: Total densitometric profile of fluorescent substrate was determined for each lane. Lower panels: Coomassie blue staining (CBS) of mitochondrial proteins was used as a loading control. (D) Chemical structure of DA29 (ClpP activator) and DA28 (negative control). (E) Effect of ClpP chemoactivation by DA29 on casein hydrolysis in the PAGE‐based fluorescent assay; DA28 was used as a negative control. For description, see panel C.
    4x Laemmli Sample Buffer Bio Rad, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 13147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Elucidating the Molecular Impact of DA29‐Induced ClpP Chemoactivation on Mitochondrial Function"

    Article Title: Elucidating the Molecular Impact of DA29‐Induced ClpP Chemoactivation on Mitochondrial Function

    Journal: The FASEB Journal

    doi: 10.1096/fj.202603398RR

    Targeted activation of endogenous mitochondrial ClpP. (A) Western blots of mitochondrial lysates (30 μg) from wild‐type and ClpP‐KO HEK293T cells were probed with antibodies against ClpP and α‐tubulin, the latter being used as a loading control. (B) Analysis of ClpP and ClpX assembly was carried out in wild‐type HEK293T mitochondria through isokinetic sucrose gradient centrifugation. Mitochondrial lysate (0.65 mg) was centrifuged on a 10%–30% gradient using the SW 60Ti rotor at 40 000 rpm for 2 h and 15 min at 4°C. Small and large mitoribosomal subunits were used as a reference of migration and were immunodecorated by antibodies against marker proteins of the mt‐SSU (mS29; HUGO protein name: DAP3) and the mt‐LSU (uL13m; HUGO protein name: MRPL13). (C) Effect of ONC201‐mediated ClpP activation on casein hydrolysis in the PAGE‐based fluorescent assay. Mitochondrial lysates (50 μg) from wild‐type and KO cells were used as a source of ClpP activity, and incubated with ONC201 at a final concentration of 100 μM. Upper panels: Milk casein isoforms (α, β and k) are depicted to the left; molecular weight markers (M; Precision Plus Protein Dual Color Standards, Bio‐Rad) are indicated to the right. Middle panels: Total densitometric profile of fluorescent substrate was determined for each lane. Lower panels: Coomassie blue staining (CBS) of mitochondrial proteins was used as a loading control. (D) Chemical structure of DA29 (ClpP activator) and DA28 (negative control). (E) Effect of ClpP chemoactivation by DA29 on casein hydrolysis in the PAGE‐based fluorescent assay; DA28 was used as a negative control. For description, see panel C.
    Figure Legend Snippet: Targeted activation of endogenous mitochondrial ClpP. (A) Western blots of mitochondrial lysates (30 μg) from wild‐type and ClpP‐KO HEK293T cells were probed with antibodies against ClpP and α‐tubulin, the latter being used as a loading control. (B) Analysis of ClpP and ClpX assembly was carried out in wild‐type HEK293T mitochondria through isokinetic sucrose gradient centrifugation. Mitochondrial lysate (0.65 mg) was centrifuged on a 10%–30% gradient using the SW 60Ti rotor at 40 000 rpm for 2 h and 15 min at 4°C. Small and large mitoribosomal subunits were used as a reference of migration and were immunodecorated by antibodies against marker proteins of the mt‐SSU (mS29; HUGO protein name: DAP3) and the mt‐LSU (uL13m; HUGO protein name: MRPL13). (C) Effect of ONC201‐mediated ClpP activation on casein hydrolysis in the PAGE‐based fluorescent assay. Mitochondrial lysates (50 μg) from wild‐type and KO cells were used as a source of ClpP activity, and incubated with ONC201 at a final concentration of 100 μM. Upper panels: Milk casein isoforms (α, β and k) are depicted to the left; molecular weight markers (M; Precision Plus Protein Dual Color Standards, Bio‐Rad) are indicated to the right. Middle panels: Total densitometric profile of fluorescent substrate was determined for each lane. Lower panels: Coomassie blue staining (CBS) of mitochondrial proteins was used as a loading control. (D) Chemical structure of DA29 (ClpP activator) and DA28 (negative control). (E) Effect of ClpP chemoactivation by DA29 on casein hydrolysis in the PAGE‐based fluorescent assay; DA28 was used as a negative control. For description, see panel C.

    Techniques Used: Activation Assay, Western Blot, Control, Analysis, Gradient Centrifugation, Migration, Marker, Fluorescence, Activity Assay, Incubation, Concentration Assay, Molecular Weight, Staining, Negative Control

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    Western Blot:

    Article Title: Novel estrogen target gene ZAS3 is overexpressed in systemic lupus erythematosus
    Article Snippet: .. Western Blotting Cells or tissue lysates were prepared using T-PER tissue protein extraction reagent (ThermoScientific) or Bio-Rad Sample buffer containing 0.5% 2-mercaptoethanol. .. Western Blotting Cells or tissue lysates were prepared using T-PER tissue protein extraction reagent (ThermoScientific) or Bio-Rad Sample buffer containing 0.5% 2-mercaptoethanol.

    Protein Extraction:

    Article Title: Novel estrogen target gene ZAS3 is overexpressed in systemic lupus erythematosus
    Article Snippet: .. Western Blotting Cells or tissue lysates were prepared using T-PER tissue protein extraction reagent (ThermoScientific) or Bio-Rad Sample buffer containing 0.5% 2-mercaptoethanol. .. Western Blotting Cells or tissue lysates were prepared using T-PER tissue protein extraction reagent (ThermoScientific) or Bio-Rad Sample buffer containing 0.5% 2-mercaptoethanol.

    Article Title: Novel estrogen target gene ZAS3 is overexpressed in systemic lupus erythematosus
    Article Snippet: .. Cells or tissue lysates were prepared using T-PER tissue protein extraction reagent (ThermoScientific) or Bio-Rad Sample buffer containing 0.5% 2-mercaptoethanol. .. Membranes were incubated with antibodies against ZAS3, ER-α (Immunotech, Marseille, France), p-ERα (Cell Signaling), and β-actin or hsp90 (Santa Cruz Biotechnologies, Santa Cruz, CA) antibodies as a loading control.

    Polyacrylamide Gel Electrophoresis:

    Article Title: FGF-7 Dictates Osteocyte Cell Processes Through Beta-Catenin Transduction
    Article Snippet: The cell images were captured using a modified confocal laser scanning microscope (CLSM, A1R MP+, Nikon, Tokyo, Japan) and analyzed with Image-Pro Plus Software 6.0. .. Protein samples were prepared by mixing one part of sample with one part of Bio-Rad Sample Buffer and then boiled at 100 °C for 5 min. Proteins were separated in 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane at 200 mA for 2 h in ice treatment. ..

    Membrane:

    Article Title: FGF-7 Dictates Osteocyte Cell Processes Through Beta-Catenin Transduction
    Article Snippet: The cell images were captured using a modified confocal laser scanning microscope (CLSM, A1R MP+, Nikon, Tokyo, Japan) and analyzed with Image-Pro Plus Software 6.0. .. Protein samples were prepared by mixing one part of sample with one part of Bio-Rad Sample Buffer and then boiled at 100 °C for 5 min. Proteins were separated in 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane at 200 mA for 2 h in ice treatment. ..



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    Targeted activation of endogenous mitochondrial ClpP. (A) Western blots of mitochondrial lysates (30 μg) from wild‐type and ClpP‐KO HEK293T cells were probed with antibodies against ClpP and α‐tubulin, the latter being used as a loading control. (B) Analysis of ClpP and ClpX assembly was carried out in wild‐type HEK293T mitochondria through isokinetic sucrose gradient centrifugation. Mitochondrial lysate (0.65 mg) was centrifuged on a 10%–30% gradient using the SW 60Ti rotor at 40 000 rpm for 2 h and 15 min at 4°C. Small and large mitoribosomal subunits were used as a reference of migration and were immunodecorated by antibodies against marker proteins of the mt‐SSU (mS29; HUGO protein name: DAP3) and the mt‐LSU (uL13m; HUGO protein name: MRPL13). (C) Effect of ONC201‐mediated ClpP activation on casein hydrolysis in the PAGE‐based fluorescent assay. Mitochondrial lysates (50 μg) from wild‐type and KO cells were used as a source of ClpP activity, and incubated with ONC201 at a final concentration of 100 μM. Upper panels: Milk casein isoforms (α, β and k) are depicted to the left; molecular weight markers (M; Precision Plus Protein Dual Color Standards, <t>Bio‐Rad)</t> are indicated to the right. Middle panels: Total densitometric profile of fluorescent substrate was determined for each lane. Lower panels: Coomassie blue staining (CBS) of mitochondrial proteins was used as a loading control. (D) Chemical structure of DA29 (ClpP activator) and DA28 (negative control). (E) Effect of ClpP chemoactivation by DA29 on casein hydrolysis in the PAGE‐based fluorescent assay; DA28 was used as a negative control. For description, see panel C.
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    Image Search Results


    Targeted activation of endogenous mitochondrial ClpP. (A) Western blots of mitochondrial lysates (30 μg) from wild‐type and ClpP‐KO HEK293T cells were probed with antibodies against ClpP and α‐tubulin, the latter being used as a loading control. (B) Analysis of ClpP and ClpX assembly was carried out in wild‐type HEK293T mitochondria through isokinetic sucrose gradient centrifugation. Mitochondrial lysate (0.65 mg) was centrifuged on a 10%–30% gradient using the SW 60Ti rotor at 40 000 rpm for 2 h and 15 min at 4°C. Small and large mitoribosomal subunits were used as a reference of migration and were immunodecorated by antibodies against marker proteins of the mt‐SSU (mS29; HUGO protein name: DAP3) and the mt‐LSU (uL13m; HUGO protein name: MRPL13). (C) Effect of ONC201‐mediated ClpP activation on casein hydrolysis in the PAGE‐based fluorescent assay. Mitochondrial lysates (50 μg) from wild‐type and KO cells were used as a source of ClpP activity, and incubated with ONC201 at a final concentration of 100 μM. Upper panels: Milk casein isoforms (α, β and k) are depicted to the left; molecular weight markers (M; Precision Plus Protein Dual Color Standards, Bio‐Rad) are indicated to the right. Middle panels: Total densitometric profile of fluorescent substrate was determined for each lane. Lower panels: Coomassie blue staining (CBS) of mitochondrial proteins was used as a loading control. (D) Chemical structure of DA29 (ClpP activator) and DA28 (negative control). (E) Effect of ClpP chemoactivation by DA29 on casein hydrolysis in the PAGE‐based fluorescent assay; DA28 was used as a negative control. For description, see panel C.

    Journal: The FASEB Journal

    Article Title: Elucidating the Molecular Impact of DA29‐Induced ClpP Chemoactivation on Mitochondrial Function

    doi: 10.1096/fj.202603398RR

    Figure Lengend Snippet: Targeted activation of endogenous mitochondrial ClpP. (A) Western blots of mitochondrial lysates (30 μg) from wild‐type and ClpP‐KO HEK293T cells were probed with antibodies against ClpP and α‐tubulin, the latter being used as a loading control. (B) Analysis of ClpP and ClpX assembly was carried out in wild‐type HEK293T mitochondria through isokinetic sucrose gradient centrifugation. Mitochondrial lysate (0.65 mg) was centrifuged on a 10%–30% gradient using the SW 60Ti rotor at 40 000 rpm for 2 h and 15 min at 4°C. Small and large mitoribosomal subunits were used as a reference of migration and were immunodecorated by antibodies against marker proteins of the mt‐SSU (mS29; HUGO protein name: DAP3) and the mt‐LSU (uL13m; HUGO protein name: MRPL13). (C) Effect of ONC201‐mediated ClpP activation on casein hydrolysis in the PAGE‐based fluorescent assay. Mitochondrial lysates (50 μg) from wild‐type and KO cells were used as a source of ClpP activity, and incubated with ONC201 at a final concentration of 100 μM. Upper panels: Milk casein isoforms (α, β and k) are depicted to the left; molecular weight markers (M; Precision Plus Protein Dual Color Standards, Bio‐Rad) are indicated to the right. Middle panels: Total densitometric profile of fluorescent substrate was determined for each lane. Lower panels: Coomassie blue staining (CBS) of mitochondrial proteins was used as a loading control. (D) Chemical structure of DA29 (ClpP activator) and DA28 (negative control). (E) Effect of ClpP chemoactivation by DA29 on casein hydrolysis in the PAGE‐based fluorescent assay; DA28 was used as a negative control. For description, see panel C.

    Article Snippet: Whole‐cell proteins (20‐30 μg), mitochondrial lysates (30 μg) or sucrose gradient fractions (10 μL) were denatured in 4X Laemmli sample buffer (Bio‐Rad) complemented with 10% 2‐mercaptoethanol, separated by SDS‐PAGE and subjected to immunoblotting as described in [ ], using the following primary antibodies: ClpP (#A306224, Antibodies.com), ClpX (#ab168338, Abcam), total OXPHOS cocktail (#ab110413, Abcam), HA‐tag (#3724, Cell Signaling Technology), uL13m (#16241–1‐AP, Proteintech), VDAC1 (#ab186321, Abcam), mS29 (# MA141279 , Thermo Fisher Scientific), α‐tubulin (#T6074, Sigma‐Aldrich) and β‐actin (#A2066, Sigma‐Aldrich).

    Techniques: Activation Assay, Western Blot, Control, Analysis, Gradient Centrifugation, Migration, Marker, Fluorescence, Activity Assay, Incubation, Concentration Assay, Molecular Weight, Staining, Negative Control