RGCs were loaded with radio immunoprecipitation assay buffer. Lysates were collected by centrifugation at 12 000 rpm for 5 min at 4 °C. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for western blotting. Membranes were blocked for 1 h at RT in 5% nonfat dry milk, then incubated with polyclonal rabbit anti-parkin (1 : 1000; Abcam, USA), monoclonal rabbit anti-Bax (1 : 1000; Abcam), polyclonal rabbit anti-Bcl-2 (1 : 500; Abcam), monoclonal rabbit anti-LC3 (1 : 2000; Abcam), polyclonal rabbit anti-LAMP1 (1 : 1000; Abcam), polyclonal rabbit anti-optineurin (1 : 200; Abcam) and polyclonal rabbit anti-GAPDH (1 : 2000; Weiao Biotechnology Co, Shanghai, China) in primary antibody dilution (Weiao Biotechnology Co) at 4 °C overnight. After washing with TBST, membranes were incubated with secondary antibodies (1 : 2000; Jackson). Blots were visualized using enhanced chemiluminescence reagents (Weiao Biotechnology Co). Chemiluminescent images were captured on X-ray film using a developing and fixing solution in a dark room and at last analyzed with Image J (National Institutes of Health, USA).
Polyclonal rabbit anti parkin
Manufactured by Abcam
Sourced in China
Polyclonal rabbit anti-parkin is a primary antibody produced in rabbits that specifically recognizes the parkin protein. Parkin is an E3 ubiquitin ligase involved in the ubiquitin-proteasome system. This antibody can be used in various immunoassays to detect and study the parkin protein.
Automatically generated - may contain errors
Lab products found in correlation
Polyclonal rabbit anti optineurin, by Abcam (5 mentions)
Monoclonal rabbit anti lc3, by Abcam (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Polyclonal rabbit anti bcl 2, by Abcam (3 mentions)
Polyclonal rabbit anti lamp1, by Abcam (3 mentions)
Monoclonal rabbit anti bax, by Abcam (3 mentions)
Polyclonal rabbit anti gapdh, by Yesen (3 mentions)
Supersignal west femto substrate trial kit, by Thermo Fisher Scientific (3 mentions)
Peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Rabbit anti gfap polyclonal antibody, by Abcam (2 mentions)
Polyclonal rabbit anti opa1, by Abcam (2 mentions)
Monoclonal mouse anti gfap antibody, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Image station 4000mm pro, by Carestream (1 mentions)
7 protocols using polyclonal rabbit anti parkin
1
Western Blot Analysis of Neurodegeneration Markers
2
Immunofluorescence Analysis of Parkin and LC3 in RGCs
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Cell culture medium was removed and cell slides were washed with PBS three times. RGCs were fixed with 4% paraformaldehyde solution for 15 min and then permeabilized with 0.2% Triton X-100 in PBS for 20 min at RT. Cell slides were washed three times with PBS. Blocking was performed with 5% BSA for 1 h at RT. After that, RGCs were incubated with polyclonal rabbit anti-parkin (1 : 200; Abcam) and monoclonal rabbit anti-LC3 (1 : 200; Abcam) overnight at 4 °C. After washing in PBS, RGCs were incubated for 1 h at 20–37 °C with Alexa Fluor 488-conjugated goat IgG secondary antibody (1 : 200; Thermo Fisher, USA). After sequential washes, RGCs were stained using Hoechst 33342 (1 μg/ml; Thermo Fisher) and washed in PBS. Finally, slides were mounted with coverslips and examined under a fluorescence microscope (Leica, DM48).
3
Retinal Immunohistochemistry of Protein Markers
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Immunohistochemical staining of 7-μm frozen sections of full-thickness retina was prepared. Three sections per frozen block from each group (n = 3 retinas/group) were used for immunohistochemical analysis. Tissue sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature and then washed thrice with PBS. Sections were next blocked with 5% bovine serum albumin/PBS for 1 h at room temperature and then with the primary antibodies against monoclonal mouse anti-GFAP antibody (1:200; Abcam), polyclonal rabbit anti-parkin (1:200; Abcam), or polyclonal rabbit anti-optineurin (1:50; Abcam) for 16 h at 4 °C. After several washes, the tissues were incubated with Alexa Fluor 488-conjugated goat IgG secondary antibody (1:200; Life Technologies) for 1 h at room temperature and then washed with PBS. The sections were counterstained with Hoechst 33342 (1 μg/ml; Life Technologies) in PBS. Images were captured by a confocal microscopy (Leica SP8).
4
Immunostaining of Retinal Ganglion Cells
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RGCs were fixed with 4% PFA in PBS for 20 min, rinsed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature, and then washed three times with PBS. The cells were then blocked with 5% BSA/PBS for 1 h at room temperature and with the primary antibodies against polyclonal rabbit anti-Tubulin antibody (1:800; Abcam) or monoclonal mouse anti-γ-synuclein (1:400; Abcam), polyclonal rabbit anti-parkin (1:200; Abcam), or polyclonal rabbit anti- optineurin (1:50; Abcam) for 16 h at 4 °C. After several washes, the RGCs were incubated with Alexa Fluor 488-conjugated goat immunoglobulin G (IgG) secondary antibody (1:200; Life Technologies) and Fluor cy3-conjugated goat anti-mouse IgG secondary antibody (1:200; Life Technologies) for 1 h at room temperature and then were washed with PBS. The RGCs were counterstained with Hoechst 33,342 (1 μg/ml; Life Technologies) in PBS. Images were captured with a confocal microscope (Leica SP8).
5
Retinal ganglion cell protein analysis
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Retinas (n = 3 per group) were lysed in RIPA buffer (Beyotime, China) and ultrasonically smashed on ice to get homogenized solutions. RGCs (n = 3 per group) were mixed with RIPA buffer. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). After blocking with 5% non-fat dry milk at room temperature for 1 h, the membranes were incubated with polyclonal rabbit anti-OPA1 (1:1000; Abcam), polyclonal rabbit anti-GFAP antibody (1:10000; Abcam), polyclonal rabbit anti-Parkin (1:1000; Abcam), polyclonal rabbit anti-Bcl-2 (1:500; Abcam), monoclonal rabbit anti-Bax (1:1,000; Abcam), monoclonal rabbit anti-LC3 (1:2000; Abcam), polyclonal rabbit anti-LAMP1 (1:1000; Abcam) and polyclonal rabbit anti-GAPDH (1:2000; Yesen, China) in primary antibody dilution (Beyotime, China) overnight at 4°C. After the membranes were washed several times, secondary antibody peroxidase-conjugated goat anti-rabbit IgG (1:5000; Jackson) was applied. Proteins were visualized by a Kodak Image Station 4000MM PRO (Carestream, Rochester, NY, United States) using chemiluminescence detection (SuperSignal West Femto Substrate Trial Kit, Thermo Fisher). Band intensity was analyzed with Image J (National Institutes of Health).
6
Western Blot Analysis of Retinal Proteins
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Retinas (n = 4 per group) were mixed with RIPA buffer (Beyotime, China) and ultrasonically smashed to get homogenized solutions. Each sample (10 μg) was separated by polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk at room temperature for 1 h, incubated with polyclonal rabbit anti-GFAP antibody (1:10,000; Abcam), polyclonal rabbit anti-Parkin (1:1000; Abcam), polyclonal rabbit anti-optineurin (1:200; Abcam), monoclonal rabbit anti-LC3 (1:2000; Abcam), polyclonal rabbit anti-LAMP1 (1:1000; Abcam), and polyclonal rabbit anti-GAPDH (1:2000; Yesen, China) in primary antibody dilution (Beyotime, China) at 4 °C overnight. The membranes were rinsed with 1×TBST (Worthington) several times, incubated with peroxidase-conjugated goat anti-rabbit IgG (1:5000; Jackson), and developed using chemiluminescence detection (SuperSignal West Femto Substrate Trial Kit, Thermo Fisher). Chemiluminescent images were captured using a Kodak Image Station 4000 MM PRO (Carestream, Rochester, NY, USA) and analyzed with Image J (National Institutes of Health).
7
Protein Expression Analysis of Retinal Ganglion Cells
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RGCs (n = 4 per group) were mixed with RIPA buffer (Beyotime, Shanghai, China). Each sample (10 μg) was separated with polyacrylamide gel electrophoresis (PAGE) and electrotransferred on polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat dry milk at room temperature for 1 h, incubated with polyclonal rabbit anti-parkin (1:1,000; Abcam), polyclonal rabbit anti- optineurin (1:200; Abcam), polyclonal rabbit anti- Bcl-2 (1:500; Abcam), monoclonal rabbit anti- Bax (1:1,000; Abcam), polyclonal rabbit anti-OPA1 (1:1,000; Abcam), and polyclonal rabbit anti-GAPDH (1:2000; Yesen, Shanghai, China) in primary antibody dilution (Beyotime) at 4 °C overnight. The membranes were rinsed with 1X Tris-buffered saline/Tween 20 (TBST; Worthington) several times, incubated with peroxidase-conjugated goat anti-rabbit IgG (1:5,000; Jackson Laboratories, West Grove, PA), and then developed using chemiluminescence detection (SuperSignal™ West Femto Substrate Trial Kit, Thermo Fisher, Waltham, MA). Chemiluminescent images were captured using a Kodak Image Station 4000MM PRO (Carestream, Rochester, NY) and analyzed with Image J (National Institutes of Health).
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