The protocol of the western blot analysis is described in detail elsewhere [9 (link)]. The primary antibodies were as follows: calpain-1 (1 : 1000; Cambridge, MA, USA, ab108400), calpain-2 (1 : 1000; Abcam, ab126600), LAMP1 (1 : 1000; Abcam, ab24170), CTSB (1 : 1000; Millipore, 06480-1), LC3 (1 : 1000; Abcam, ab63817), Beclin-1 (1 : 1000; CST, Technology, Boston, MA, USA, #3495), P62 (1 : 1000; Abcam, ab109012), ATG5 (1 : 500; CST, #12994), TSG101 (1 : 1000; Abcam, ab125011), CD63 (1 : 1000; Abcam, ab271286), V-ATPase (1 : 500; Abcam, ab105937), VAMP7 (1 : 1000; CST, #13920), and β-actin (1 : 500; Proteintech, Chicago, USA, 66009).
Ab108400
Manufactured by Abcam
Sourced in United States
Ab108400 is a lab equipment product. It is a recombinant monoclonal antibody targeting a specific protein. The core function of this product is to serve as a detection and quantification tool for the target protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ab125011, by Abcam (1 mentions)
Ab271286, by Abcam (1 mentions)
Ab24170, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Ab109300, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab81286, by Abcam (1 mentions)
Ab7671, by Abcam (1 mentions)
Ab3583, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab4051, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Quantity one software, by Media Cybernetics (1 mentions)
Western bright ecl kit, by Bio-Rad (1 mentions)
Anti caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Antirabbit or antimouse igg, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
6 protocols using ab108400
1
Western Blot Analysis of Autophagy Markers
2
Nuclear Translocation of NF-κB Pathway
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Heart tissues from each group were used to isolate p65 in the nucleus, as well as IκBα, p-IκBα, and p65 in the cytoplasm by using a nuclear protein extraction kit. RIPA buffer was used to isolate the calpain-1 protein whose concentration was determined by BCA protein assay (Thermo Fisher, USA). During the Western blot experiment, proteins were separated with 10% SDS-PAGE gels while the target proteins were transferred to a PVDF film. After blocking with 5% skimmed milk for 1 h, they were incubated with primary antibody calpain-1 (ab108400, Abcam, UK), p-IκBα (ab109300, Abcam, UK), p65 (ab16502, Abcam, UK), β-actin (ab8226, Abcam, UK), and Histone3 (ab1791, Abcam, UK) overnight at 4°C with the dilution 1 : 2000, after which they were incubated again with the corresponding second antibody for 1 h at room temperature. After rinsing with TBST, they were placed in a gel imaging analysis system together with ECL. The reaction was performed for 3 minutes. Then, they were exposed to collect pictures via Image Lab™ software. There was a gray analysis with β-actin used as an internal reference.
3
Protein Expression Analysis of NETs
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A sample containing 80 μg of the extracted proteins from the NETs induced by PMA, S. suis or PBS was subjected to a Western blot analysis with antibodies against PGRP-S (sigma, SAB2500783), WDR5 (sigma, PLA0256), Lysozyme (abcam, ab158508), Calpain (abcam, ab108400), or MMP8 (abcam, ab81286). The expression of GAPDH was also determined as a reference control with a GAPDH antibody (Calbio, CB100127).
4
Western Blot Analysis of Cardiac Proteins
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Expression levels of leptin, caspase 3, Bax, Bcl‐2, NKA, calpain, sodium‐calcium exchanger 1, and β‐actin proteins were determined by Western blot, as described previously.17 The bands of target proteins and internal reference β‐actin were scanned and analyzed by Quantity One software (Media Cybernetics Inc). The results are shown as the ratio of integrated optical density of the target proteins/the internal control and statistically analyzed. The primary antibodies used were anti–β‐actin antibody (JC‐PA002; 1:1000; Jingcai Biology, China), anti–caspase 3 antibody (ab4051; 1:500; Abcam), anti‐Bax antibody (1:1000; Abcam), anti‐leptin antibody (ab3583; 1:1000; Abcam), anti–Bcl‐2 antibody (ab59348; 1:1000; Abcam), anti–cytochrome C antibody (ab133504; 1:1000; Abcam), anti–calpain 1 antibody (ab108400; 1:5000; Abcam), and anti‐α1 NKA antibody (ab7671; 1:1000; Abcam).
5
Protein Extraction and Western Blot Analysis
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Protein extraction from the cell pellets was performed using a total protein extraction kit (Applygen Technologies, China) in accordance with the manufacturer's protocol. The protein concentrations were determined using a BCA protein assay reagent kit (Novagen, USA). Then, 20 μg of total protein samples were loaded and separated by SDS/PAGE electrophoresis and transferred to PVDF membranes (Millipore Corporation, USA). After blocking with 5% skim milk in TBST buffer (Tris-buffered saline, 0.1% Tween 20) at room temperature for 2 h, the membranes were incubated with the primary antibody (1:5000) at 4°C for 8 h. Subsequently, the membranes were incubated with a secondary antibody (1:5000, anti-rabbit, or anti-mouse IgG, Abcam, USA). Then, the protein level on the blot was detected using the Western Bright ECL kit (Bio-Rad Laboratories, USA). The following antibodies against the target proteins were used: rabbit monoclonal anti-calpain-1 antibody (ab108400, Abcam), rabbit monoclonal anti-caspase-3 antibody (9664, Cell Signaling Technology, USA), mouse monoclonal anti-caspase-8 antibody (9496, Cell Signaling Technology), and rabbit polyclonal anti-GAPDH antibody (ab9485, Abcam). Detection of GAPDH was used as the internal control.
6
Immunohistochemical Analysis of Lens Proteins
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After deparaffinization and rehydration, the sections were boiled for 10 min in diluted with ddH 2 O from ×100 to ×1 antigen repair solution (MXB Biotechnologies, China) and blocked with 5% BSA for 1 h. Anti-Capn1 monoclonal
antibody (ab108400, ABCAM, USA, 1:100), Anti-Tgm2 polyclonal antibody (ab421, ABCAM, USA, 1:500), Anti-Camk2b polyclonal antibody (ab34703, ABCAM, USA, 1:200), Anti-Gja8 polyclonal antibody (ab222885, ABCAM, USA, 1:100); Anti-Cryab polyclonal antibody (ab5577, ABCAM, USA, 1:200), anti-Cryba1 polyclonal antibody (PA5-71690, ThermoFisher Scientific, USA, 1:500), anti-Crybb1 polyclonal antibody (bs-12582R, Bioss Antibodies, China, 1:500), and anti-Crybb3 polyclonal antibody (21009-1-AP, Proteintech Systems, USA, 1:100) were used as primary antibody. Anti-Rabbit antibody488 (A-21206, Invitrogen, USA), and anti-Rabbit antibody594 (A-21207, Invitrogen, USA) as secondary antibodies were incubated at room temperature for 2 h. Cell nuclei were counterstained with DAPI. DAB (TA-060-QHDX Ther-moFisher Scientific, USA) was used for color development followed by hematoxylin counterstaining in immunohistochemistry assay. Three mice/six lenses from each group were evaluated to compare these proteins expression of these two groups and repeated 3 times.
antibody (ab108400, ABCAM, USA, 1:100), Anti-Tgm2 polyclonal antibody (ab421, ABCAM, USA, 1:500), Anti-Camk2b polyclonal antibody (ab34703, ABCAM, USA, 1:200), Anti-Gja8 polyclonal antibody (ab222885, ABCAM, USA, 1:100); Anti-Cryab polyclonal antibody (ab5577, ABCAM, USA, 1:200), anti-Cryba1 polyclonal antibody (PA5-71690, ThermoFisher Scientific, USA, 1:500), anti-Crybb1 polyclonal antibody (bs-12582R, Bioss Antibodies, China, 1:500), and anti-Crybb3 polyclonal antibody (21009-1-AP, Proteintech Systems, USA, 1:100) were used as primary antibody. Anti-Rabbit antibody488 (A-21206, Invitrogen, USA), and anti-Rabbit antibody594 (A-21207, Invitrogen, USA) as secondary antibodies were incubated at room temperature for 2 h. Cell nuclei were counterstained with DAPI. DAB (TA-060-QHDX Ther-moFisher Scientific, USA) was used for color development followed by hematoxylin counterstaining in immunohistochemistry assay. Three mice/six lenses from each group were evaluated to compare these proteins expression of these two groups and repeated 3 times.
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