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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 11850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: Allosteric Biosensors Unravel GTPase-Effector Feedback
    Article Snippet: .. The supernatant was mixed with 4X Laemmli sample buffer (Bio-Rad) with 10% 2-mercaptoethanol (Sigma), boiled for 5 mins and stored at -20°C for Western blotting. .. Anti-FLAG M2 agarose beads (Sigma) were blocked with 1% BSA in IP buffer overnight at 4°C.

    Article Title: Cell Cycle Control of Nuclear Metabolism Couples Phosphatidylinositol Signaling to Histone Methylation.
    Article Snippet: The concentrations of the cytosolic and hromatome extracts were determined using the Pierce BCA rotein Assay Kit (Thermo Scientific; #PIER23225). .. .13 Western Blot he samples were combined with 4X Laemmli sample buffer Bio-Rad; #1610747) and heated to 95◦C for 3 min. ..

    other:

    Article Title: Systemic Propagation of STING Signalling via Generation of Large Extracellular Vesicles.
    Article Snippet: Cells were cultured in DMEM (Life Technologies, 1-965-118) supplemented with 10% FBS (Life Technologies, 16140- 71) and 1% penicillin-streptomycin (Life Technologies, 15-140- 22) and were regularly tested for mycoplasma (Applied Biologial Materials, G328).

    Incubation:

    Article Title: Development of a PTEN -siRNA activated scaffold to promote axonal regrowth following spinal cord injury
    Article Snippet: Next, the total protein content was measured using a PierceTM BCA Protein Assay Kit (ThermoFisher Scientific, Ireland) according to the manufacturer's instructions. .. Thereafter, 15 μg total protein was incubated at 95 °C with 4x Laemmli sample loading buffer (BioRad) before loading into a gradient gel (Mini-PROTEAN TGX Gels 4–20 %, BioRad). .. Proteins were separated by electrophoresis and subsequently transferred to a polyvinylidene fluoride or polyvinylidene difluoride (Trans-Blot Turbo Transfer - 0.2 μm PVDF, BioRad) membrane and blocked with 5 % (w/v) fat-free milk in 0.1 % (v/v) tris-buffered saline (TBS)-Tween 20 (TBST) (Sigma-Aldrich, Ireland).



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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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    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