Total protein was extracted from liver tissues using the RIPA buffer (sigma, R0278) and the Protease and phosphatase inhibitor cocktail (Thermo Scientific). After the concentration being quantified by BCA protein assay kit, equal amounts of protein from animals of the same group were pooled. Western blotting was performed as described previously27 (link). Rabbit anti-light chain 3 (LC3B; 1:1000, Abcam), rabbit anti-Caspase3 (1:1000, Cell signaling Technology), rabbit anti-SQSTM1/p62 (8025, 1:1000, Cell signaling Technology) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000, Cell signaling Technology) were used as primary antibodies. Goat polyclonal antibody to rabbit IgG (1:5000) was used as secondary antibody. The membranes were probed using enhanced chemiluminescence western blotting substrate (GE Healthcare) and exposed to high sensitivity film (GE Healthcare) or digitalized with Fusion FX7 (Labtech International Ltd, Heathfield, United Kingdom). The signal intensity of each protein band was quantified using Gel Analyzer Module provided by ImageJ (National Institutes of Health, Bethesda, MD). All Western blots were repeated 3 times.
Rabbit anti glyceraldehyde 3 phosphate dehydrogenase gapdh
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a primary antibody raised in rabbits that recognizes the GAPDH protein. GAPDH is an enzyme involved in the glycolysis pathway, catalyzing the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence western blotting substrate, by GE Healthcare (1 mentions)
High sensitivity film, by GE Healthcare (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Glutamate, by Merck Group (1 mentions)
Mouse anti vdac1, by Abcam (1 mentions)
Mk 801, by Bio-Techne (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Mouse anti actin, by Merck Group (1 mentions)
Mouse anti human tdp 43, by Novus Biologicals (1 mentions)
Rabbit anti tdp 43, by Proteintech (1 mentions)
Rabbit anti sod1, by Santa Cruz Biotechnology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Anti omi htra2, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using rabbit anti glyceraldehyde 3 phosphate dehydrogenase gapdh
1
Evaluating Liver Protein Expression
2
Western Blot Analysis of IL-4Rα in Neuropathic Pain
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The ipsilateral dorsal part of the lumbar spinal cords was collected from animals on the 14th day after CCI. Spinal tissue and PC12 cells were lysed in radioimmunoprecipitation assay buffer and centrifuged at 14,000 × g for 30 min at 4°C. Proteins were electrophoresed in 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Merck Millipore, Billerica, MA). The membranes were blocked with 5% bovine serum albumin for 1 h and then incubated with rabbitpolyclonal anti-IL-4Rα (1:200; Santa Cruz Biotechnology) or rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000; Cell Signaling Technology) for 24 h at 4°C. The membrane was washed and incubated with horseradish peroxidase-conjugated anti-rabbit antibody (1:4000; ConWin Biotech, Beijing, China) for 2 h at room temperature. Immunoreactive bands were visualized using super ECL detection reagent (Merck Millipore). ImageJ software (National Institutes of Health, Bethesda, MD) was used to analyze band densities.
3
Immunoblotting Analysis of Neurodegeneration Markers
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Tissues or culture motor neurons were lysed with 1XCell Lysis Buffer (Cell Signaling Technology, Beverly, MA, USA), plus 1 mM phenylmethylsulfonyl fluoride (Sigma) and Protease Inhibitor Cocktail (Sigma). Equal amounts of total protein extract (10 ug) were resolved by sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P (Millipore). Following blocking with 10% non-fat dry milk, primary and secondary antibodies were applied and the blots developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore). Frozen human thoracic spinal cord tissues from 6 age-matched normal (age 58 3.8; sex, M:F = 5:1) and 8 patients with sALS (age 57 3.9; sex, M:F = 6:2) were obtained from NICHD (The Eunice Kennedy Shriver National Institute of Child Health and Human Development) Brain and Tissue Bank. B6SJL-Tg (SOD1*G93A) 1Gur/J (SOD1G93A) mice and C57BL/6-Tg (Prnp-TARDBP*M337V) 4Ptrc/J (TDP-43M337V) mice were obtained from the Jackson Laboratory. Primary antibodies used included mouse anti-RHOT1 for detecting Miro1 (Abnova), mouse anti-VDAC1 (Abcam), rabbit anti-TDP-43 (Proteintech), mouse anti-human TDP-43 (Novus), rabbit anti-SOD1 (Santa Cruz), mouse anti-actin (Millipore, Billerica, MA) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling). Glutamate (Sigma) and MK-801 (Tocris) were also obtained.
4
Western Blot Protocol for Protein Quantification
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Protein lysates from tissues were prepared as previously described [13 (link)], and protein concentrations of lysates were determined using a BCA Protein Assay Kit (#23225; Thermo Scientific Pierce, Carlsbad, CA, USA). Fifty µg/lane of each sample were run on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels, depending of the target proteins, and then were electrotransferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies anti-Omi/HtrA2 (#2176; 1:1000; Cell Signaling Technology, Danvers, MA, USA) or rabbit anti-glyceraldehyde-3-phosphate dehydrogenase/GAPDH (#2118; 1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, followed by incubation with the matched secondary antibodies for 1h at room temperature. The density of the target protein bands was measured using ImageJ software, and GAPDH was used as a protein loading control.
5
Western Blot Protocol for Protein Analysis
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Cells were collected and lysed with 1× SDS gel-loading buffer. Proteins (30 μg of total protein per well) were separated by SDS-PAGE. The proteins were transferred to a nitrocellulose Amersham Hybond-ECL membrane (Thermo Fisher Scientific, Waltham, MA, USA). The membrane was blocked with a solution of 5% dried milk, 0.1% Tween-20 in PBS during 30 min at RT, and incubated with sheep anti-VIII (1:250) (Abcam, Cambridge, MA, USA), mouse anti-actin JLA20-s antibodies (1:500) (DSHB, Iowa City, IA, USA), or rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000) (Cell Signaling, Danvers, MA, USA) in 1% milk, 0.1% Tween-20 in PBS overnight at 4 °C. The membranes were washed three times for 5 min with 0.1% Tween-20 in PBS, and incubated with solutions of corresponding peroxidase-conjugated secondary antibodies: donkey anti-sheep, goat anti-rabbit, and goat anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA, USA), diluted in 1% milk, 0.1% Tween-20 in PBS for 1 h at RT. After washing the membranes three times, they were analyzed using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and Chemidoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).
6
Western Blot Analysis of Neuronal Proteins
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Western blot analyses in the brain were carried out as previously described (Yuan et al., 2016). Primary antibodies included rabbit anti-CEND1(1:1000; Cat# ab113076; Abcam), mouse anti-tubulin (1:1000, Cat# ab7291; Abcam), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000; Cat# 5174; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-Notch1 (1:1000; Cat# 3608; Cell Signaling Technology), rabbit anti-cyclin D1 (1:1000; Cat# 2978; Cell Signaling Technology), and rabbit anti-p21 (1:1000; Cat# 2947; Cell Signaling Technology), which were incubated overnight at 4°C. After washing, membranes were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:1000; Cat# 7074; Cell Signaling Technology) for 1 hour at room temperature and then reacted with an enhanced chemiluminescence substrate (Pierce, Rockford, IL, USA). Chemiluminescence was detected using Quantity One image software (Bio-Rad, Hercules, CA, USA), and relative intensities were calculated using Gel-Pro Analyzer software (Media Cybernetics, Bethesda, MD, USA).
7
Antibodies for VAPB and PTPIP51 Proteins
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Rat and rabbit antibodies to VAPB and PTPIP51 have been described previously and were generated by immunization with GST‐VAPB(1–220) and GST‐PTPIP51(36–470) 5 . Rabbit PTPIP51 antibody (FAM82A2) was from Atlas Antibodies. Rabbit anti‐haemagglutinin (HA), mouse anti‐α‐tubulin (DM1A) and rabbit anti‐mitofusin‐2 were from Sigma. Mouse anti‐myc (9B11) and rabbit anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) were from Cell Signaling. Rabbit anti‐FUS (NB100‐565) was from Novus Biologicals. Rabbit anti‐TOM20 was from Santa Cruz Biotechnology and mouse anti‐PDI (RL77) was from Affinity Bioreagents. Antibodies to total and ser9 phosphorylated (inactive) GSK‐3β were from BD Transduction Labs (mouse 610201) and Cell Signalling (rabbit 9336), respectively. Rabbit anti‐GFP (Ab290) was from Abcam. GSK‐3β inhibitors AR‐A014418 and CT99021 were from Abcam and Cayman, respectively, and made up as 1 mM or 100 μM stocks in DMSO; KCN was from Sigma and made up as a 1 M stock in water.
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