The following primary antibodies were used: Drp1 (611113; BD Biosciences), Opa1 (612607; BD Biosciences), PDH (ab110333; Abcam), GAPDH (MA5-15738; Thermo), Tim44 (612582; BD Transduction Laboratories), Tim23 (611223; BD Transduction Laboratories), Tom20 (sc-11415; Santa Cruz Biotechnology), mitofusin 1 (ab57602; Abcam), mitofusin 2 (ab56889; Abcam), HA (600-401-384; Rockland), VDAC (4866; Cell Signaling Technology), HSP60 (12165; Cell Signaling Technology), E-cadherin (14472; Cell Signaling Technology), p62 (GP-62C; Progen), ubiquitin (z0458; DAKO), phospho-p62 (serine 351) (PM074; MBL International), LC3 (PM036; MBL) and Keap1 (10503-2-AP; Proteintech). The following secondary antibodies were purchased from Invitrogen: Alexa 488 anti-Rabbit IgG (A21206), Alexa 488 anti-Mouse IgG (A21202), Alexa 488 anti-guinia pig IgG (A11073), Alexa 568 anti-mouse IgG (A10037), Alexa 647 anti-mouse IgG (A31571) and Alexa 647 anti-guinia pig IgG (A21450).
Ab110333
Manufactured by Abcam
Ab110333 is a protein assay kit that can be used to quantify the total protein content of a sample. The kit provides the necessary reagents and buffers to perform the assay.
Automatically generated - may contain errors
Lab products found in correlation
Cryostat microtome, by Leica (3 mentions)
Ab150176, by Abcam (3 mentions)
Ab150120, by Abcam (3 mentions)
Fluoromount, by Diagnostic BioSystems (3 mentions)
Ab13970, by Abcam (3 mentions)
Ab56889, by Abcam (2 mentions)
Ab186735, by Abcam (2 mentions)
Ab52950, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ubiquitin, by Agilent Technologies (1 mentions)
Ab57602, by Abcam (1 mentions)
Keap1, by Proteintech (1 mentions)
Tom20, by Santa Cruz Biotechnology (1 mentions)
Alexa 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa 568 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
P62 gp62 c, by Progen Biotechnik (1 mentions)
Tim23, by BD (1 mentions)
Tim 44, by BD (1 mentions)
Hsp60, by Cell Signaling Technology (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
7 protocols using ab110333
1
Mitochondrial Dynamics and Protein Quantification
2
Immunofluorescence Staining of Cultured Cells
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Coverslips were seeded with cultured cells at 5 × 104 cells/ml in 500 μL of DMEM culture medium, then fixed for 15 min at room temperature with 4 % paraformaldehyde. After permeabilization with 0.25 % Triton X-100 in PBS for 10 min, cells were blocked with 1 % bovine serum albumin in PBST for 30 min. The primary antibody was first added to the samples and incubated overnight at 4 °C. Following washing with PBS, the secondary antibody in 1 % BSA/PBST was then incubated at room temperature for 1 h. Discard the antibody and wash the slides. The nucleus was stained with Hoechst33258 (94403, Sigma) for 5 min, and then the coverslip was mounted with a drop of fluorescent mounting medium (S3023, Dako). Store the slides in the dark at −20 °C. The secondary antibodies are as follows: Donkey anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A21207, Invitrogen), Donkey anti-mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A21202, Invitrogen). The primary antibodies were applied as follows: anti-PDH (1:2000, ab110333, Abcam) and anti-TOM20 (1:1000, ab186735, Abcam), anti-hSOD1 (1:1000, ab52950, Abcam), and anti-HMGB1(1:1000, ab18256, Abcam).
3
Immunohistochemistry for Mouse Brain Sections
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For staining mouse DLS and HPC sections, mice were transcardially perfused with 10% formalin and brains were extracted. Brains were postfixed in 10% formalin overnight at 4°C and cryoprotected in 30% sucrose for 48-72 hrs. 40 µm thick coronal sections were obtained using a cryostat microtome (Leica) and preserved in 0.01% sodium azide + PBS at 4°C until use.
Immunohistochemistry was performed using previously published techniques 16, 29 (link) . Briefly, sections were washed 3x for 10 min in 1X TBS, then blocked for 1 hr at RT in 1X TBS solution containing 5% NGS + 0.25% Triton-X-100. Sections were incubated overnight at 4°C in primary antibodies diluted in blocking solution. The following primary antibodies were used: chicken anti-GFP (1:2000; Abcam ab13970) and mouse anti-pyruvate dehydrogenase (PDH) (1:1000, Abcam ab110333). The following day sections were washed 3x for 10 min each in 1X TBS and incubated with appropriate secondary antibodies in blocking solution for 2 hr at RT. The following secondary antibodies were used: Alexa-488 goat anti-chicken (1:2000; Abcam ab150176) and Alexa-594 goat anti-mouse (1:2000; Abcam ab150120). Sections were rinsed 3x for 10 min in 1X TBS and then mounted on microscope slides in Fluoromount (Diagnostic Biosystems; K024) for imaging.
Immunohistochemistry was performed using previously published techniques 16, 29 (link) . Briefly, sections were washed 3x for 10 min in 1X TBS, then blocked for 1 hr at RT in 1X TBS solution containing 5% NGS + 0.25% Triton-X-100. Sections were incubated overnight at 4°C in primary antibodies diluted in blocking solution. The following primary antibodies were used: chicken anti-GFP (1:2000; Abcam ab13970) and mouse anti-pyruvate dehydrogenase (PDH) (1:1000, Abcam ab110333). The following day sections were washed 3x for 10 min each in 1X TBS and incubated with appropriate secondary antibodies in blocking solution for 2 hr at RT. The following secondary antibodies were used: Alexa-488 goat anti-chicken (1:2000; Abcam ab150176) and Alexa-594 goat anti-mouse (1:2000; Abcam ab150120). Sections were rinsed 3x for 10 min in 1X TBS and then mounted on microscope slides in Fluoromount (Diagnostic Biosystems; K024) for imaging.
4
Western Blot Analysis of Cellular Markers
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As previously described, tissue was homogenized, and cells were broken in the RIPA lysis buffer (BP571-5, Fisher) [30 (link)]. The protein concentrations were measured by using a BCA protein assay kit (23225, ThermoFisher). Afterwards, the homogenates were electrophoretically separated using SDS-PAGE. Proteins were separated by electrophoresis and transferred to PVDF membranes, which were blocked in the blocking buffer (5 % milk in PBST) for an hour. A primary antibody was then incubated overnight at 4 °C on the membrane, followed by a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. An ECL kit was used to visualize the antigen-antibody complexes (1705061, Bio-Rad). The primary antibodies used were as follows: anti-hSOD1 (1:1000, ab52950, Abcam), anti-A5C3 (1:500, MM0070-3-P, Medimabs), anti-SIRT1 (1:1000, ab11034, Abcam), anti-SIRT6(1:1000, ab191385, Abcam), anti-P53 (1:1000, ab26, Abcam), anti-P16INK4A (1:1000, sc166760, Santa Cruz), anti-MFN2 (1:1000, ab56889, Abcam), anti-PGC-1α (1:500, ab54481, Abcam), anti-TOM20 (1:1000, ab186735, Abcam), anti-PDH (1:1000, ab110333, Abcam), anti-STX17 (1:500, ab229646, Abcam), anti-β-actin (1:5000, sc47778, Santa Cruz) and Histone H1 (1:1000, sc8030, Santa Cruz). Histone H1 (for the nuclear fraction) and β-action (for total protein and cytoplasmic fraction) were used as loading controls.
5
Mitochondrial Protein Immunoblotting Protocol
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Mitochondrial suspensions or MDV fractions were collected in SDS‐PAGE buffer (6X Laemmli buffer), vortexed, boiled at 95°C for 5 minutes, and stored at −80°C. Equal volumes of samples were loaded onto a stain‐free gel (TGX Stain‐Free FastCast, 10%, 1.5 mm thick) and electrophoresis was performed at 150 V for 1 hour. PVDF membranes were activated in 100% methanol for 20 seconds, transferred to water for 2 minutes, and left in cold transfer buffer until use. Gels were activated using UV light for 45 seconds in a ChemiDoc Touch imaging system (Bio‐Rad) at high power and 365 nm wavelength. Proteins were transferred onto the membrane using a Trans‐Blot Turbo transfer system (Bio‐Rad) at 1.3 A and up to 25 V for 10 min. Stain‐free images were taken by exposing membranes to UV light using the above‐mentioned settings. Membranes were probed with an anti‐PDHE2/E3bp antibody (ab110333, Abcam) using Snap ID 2.0. Bands were visualized via chemiluminescence using Clarity Western ECL substrate (Bio‐Rad) and imaged under a ChemiDoc Touch imaging system (Bio‐Rad).
6
Immunohistochemistry for Mouse Brain Sections
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For staining mouse DLS and HPC sections, mice were transcardially perfused with 10% formalin and brains were extracted. Brains were postfixed in 10% formalin overnight at 4°C and cryoprotected in 30% sucrose for 48-72 hrs. 40 µm thick coronal sections were obtained using a cryostat microtome (Leica) and preserved in 0.01% sodium azide + PBS at 4°C until use.
Immunohistochemistry was performed using previously published techniques 16, 29 (link) . Briefly, sections were washed 3x for 10 min in 1X TBS, then blocked for 1 hr at RT in 1X TBS solution containing 5% NGS + 0.25% Triton-X-100. Sections were incubated overnight at 4°C in primary antibodies diluted in blocking solution. The following primary antibodies were used: chicken anti-GFP (1:2000; Abcam ab13970) and mouse anti-pyruvate dehydrogenase (PDH) (1:1000, Abcam ab110333). The following day sections were washed 3x for 10 min each in 1X TBS and incubated with appropriate secondary antibodies in blocking solution for 2 hr at RT. The following secondary antibodies were used: Alexa-488 goat anti-chicken (1:2000; Abcam ab150176) and Alexa-594 goat anti-mouse (1:2000; Abcam ab150120). Sections were rinsed 3x for 10 min in 1X TBS and then mounted on microscope slides in Fluoromount (Diagnostic Biosystems; K024) for imaging.
Immunohistochemistry was performed using previously published techniques 16, 29 (link) . Briefly, sections were washed 3x for 10 min in 1X TBS, then blocked for 1 hr at RT in 1X TBS solution containing 5% NGS + 0.25% Triton-X-100. Sections were incubated overnight at 4°C in primary antibodies diluted in blocking solution. The following primary antibodies were used: chicken anti-GFP (1:2000; Abcam ab13970) and mouse anti-pyruvate dehydrogenase (PDH) (1:1000, Abcam ab110333). The following day sections were washed 3x for 10 min each in 1X TBS and incubated with appropriate secondary antibodies in blocking solution for 2 hr at RT. The following secondary antibodies were used: Alexa-488 goat anti-chicken (1:2000; Abcam ab150176) and Alexa-594 goat anti-mouse (1:2000; Abcam ab150120). Sections were rinsed 3x for 10 min in 1X TBS and then mounted on microscope slides in Fluoromount (Diagnostic Biosystems; K024) for imaging.
7
Immunofluorescent Staining of Mouse Brain Sections
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For staining mouse DLS and HPC sections, mice were transcardially perfused with 10% formalin and brains were extracted. Brains were post xed in 10% formalin overnight at 4°C and cryoprotected in 30% sucrose for 48-72 hrs. 40 µm thick coronal sections were obtained using a cryostat microtome (Leica) and preserved in 0.01% sodium azide + PBS at 4°C until use. Immunohistochemistry was performed using previously published techniques16,29. Brie y, sections were washed 3x for 10 min in 1X TBS, then blocked for 1 hr at RT in 1X TBS solution containing 5% NGS + 0.25% Triton-X-100. Sections were incubated overnight at 4°C in primary antibodies diluted in blocking solution. The following primary antibodies were used: chicken anti-GFP (1:2000; Abcam ab13970) and mouse anti-pyruvate dehydrogenase (PDH) (1:1000, Abcam ab110333). The following day sections were washed 3x for 10 min each in 1X TBS and incubated with appropriate secondary antibodies in blocking solution for 2 hr at RT. The following secondary antibodies were used: Alexa-488 goat anti-chicken (1:2000; Abcam ab150176) and Alexa-594 goat anti-mouse (1:2000; Abcam ab150120). Sections were rinsed 3x for 10 min in 1X TBS and then mounted on microscope slides in Fluoromount (Diagnostic Biosystems; K024) for imaging.
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