The protein extracts prepared after sample collection were detected by WB. The oxidative stress–associated protein nuclear factor–erythroid 2-related factor-2 (Nrf-2; ab92946, Abcam), lipid peroxidation–associated protein malondialdehyde (MDA; ab27642, Abcam), mitochondrial injury–associated protein cytochrome c oxidase IV (COXIV; ab202554, Abcam), heat shock protein 60 (HSP60; ab190828, Abcam), system Xc−–associated protein solute carrier family 7, member 11 (SLC7A11; ab175186, Abcam), glutathione peroxidase-4 (GPX4; ET1706-45, HUABIO), ferroptosis-associated protein ferroportin-1 (FPN1; ab239511, Abcam), and divalent metal transporter 1 (DMT1; ab157208, Abcam) were used, with β-actin (AF7018, Affinity, England) as the control. Goat anti-rabbit IgG (Biosharp, China) was used as the secondary antibody. A western blotting ECL kit (ZETA-Life, San Francisco, CA, USA) was used. After the detection step, protein bands were observed and photographed using an automated chemiluminescence image analysis system (Tanon, Shanghai, China), and various parameters were quantified using ImageJ software (National Institutes of Health, DC, USA). The procedure was repeated three times before statistical analysis using SPSS 26.0.
Ab190828
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab190828 is a monoclonal antibody that recognizes human Calnexin. Calnexin is a molecular chaperone that is involved in the folding and quality control of glycoproteins in the endoplasmic reticulum.
Automatically generated - may contain errors
Lab products found in correlation
Ab14734, by Abcam (2 mentions)
A4700, by Merck Group (2 mentions)
X omat film, by Kodak (2 mentions)
Ab239511, by Abcam (1 mentions)
Ab157208, by Abcam (1 mentions)
Ab27642, by Abcam (1 mentions)
Ab202554, by Abcam (1 mentions)
Ab92946, by Abcam (1 mentions)
Ab175186, by Abcam (1 mentions)
Et1706 45, by Huabio (1 mentions)
Goat anti rabbit igg, by Biosharp (1 mentions)
Af7018, by Affinity Biosciences (1 mentions)
β actin, by Affinity Biosciences (1 mentions)
Ab186735, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab73015, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Pa5 27137, by Thermo Fisher Scientific (1 mentions)
Ab56889, by Abcam (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
6 protocols using ab190828
1
Protein Expression Analysis of Oxidative Stress
2
Immunofluorescence Imaging of Mitochondrial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% paraformaldehyde. Then, 0.3% Triton X-100 was added to the cells to control penetrability. The cells were then incubated with primary antibodies, such as anti-PINK1 (ab216144, 1:1000, Acam, USA), anti-Tom20 (ab186735, 1:1000, Abcam, USA), anti-Parkin (ab73015, 1:1000, Abcam, USA) and anti-ASCL4 (PA5-27137, 1:1000, Invitrogen, USA), anti-cyto C (ab133504, 1:5000, Abcam USA), and Hsp60 (ab190828, 1:1000, Abcam, USA) in the shade at room temperature for 1.5 h, followed by incubation with secondary goat-anti-rabbit antibodies (ab6721, 1:2000, Abcam, USA) for 1 h. 4´,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei.
3
Protein Expression Analysis by Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell lysates were boiled at 100 °C for 5 min in 1.5× sample buffer (1.5 M Tris pH 6.8; 15% glycerol; 3% SDS; 7.5% β-mercaptoethanol; 0.0375% bromophenol blue). Gel electrophoresis was performed using 9% polyacrylamide gels. The gels were blotted onto PVDF membranes, which were then blocked for 1 h at room temperature with 5% (w/v) non-fat dried milk in PBS with 0.05% Tween 20. The membranes were then incubated overnight at 4 °C with the primary antibodies diluted in blocking solution as follows: VDAC1 (1:1000; Ab14734; Abcam, Cambridge, UK), HSP60 (1:2000; Ab190828; Abcam) and actin (1:1000, A4700, Merck, Sigma-Aldrich, Burlington, VT, USA). To visualize Western blots, HRP-based secondary antibodies were used, followed by chemiluminescent detection on Kodak X-Omat film.
4
Western Blot Analysis of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell lysates were boiled at 100°Cfor 5 min in 1.5× sample buffer (1.5 M Tris pH 6.8; 15% Glycerol; 3% SDS; 7.5% β‐mercaptoethanol; 0.0375% bromophenol blue). Gel electrophoresis was performed using 9% polyacrylamide gels. The gels were blotted onto PVDF membranes, which were then blocked for 1 h at room temperature with 5% (w/v) non‐fat dried milk in PBS with 0.05% Tween 20. The membranes were then incubated overnight at 4°Cwith the primary antibodies diluted in blocking solution as follows: Mfn2 (1:2,000; Ab56889; Abcam), Mfn1 (1:250; sc‐50330; Santa Cruz Biotechnology), Drp1 (1:1,000, 611112, BD Biosciences) VDAC1 (1:1,000; Ab14734; Abcam), HSP60 (1:2,000; Ab190828; Abcam), MCU (1:1,000; #14997; Cell Signaling Technology), p‐eIF2alpha (1:200; sc‐101670; Santa Cruz Biotechnology), eIF2alpha (1:1,000; ab242148; Abcam), myc (1:500, M4439, Sigma), GFP (1:1,000, A11122, Life Technologies), RFP (1:500, 600‐401‐379, Rockland), GFAP (1:500, Z0334, Dako), and actin (1:10,000, A4700, Sigma). To visualize Western blots, HRP‐based secondary antibodies were used followed by chemiluminescent detection on Kodak X‐Omat film.
5
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, for western blot analysis, 50 μg of proteins were loaded onto a 12% poly-acrylamide gel Mini- PROTEAN® TGXTM (BIO-RAD, Milan, Italy). Electro-transfer to nitrocellulose membrane was obtained through Trans- Blot® TurboTM (BIO-RAD), using Trans-Blot® SE Semi-Dry Transfer Cell (BIO-RAD) [20 (link), 21 (link)]. Membranes were blocked in Odyssey Blocking Buffer (Licor, Milan, Italy), according to the manufacturer’s protocol. After blocking, membranes were washed three times in PBS for 5 min and incubated with primary antibodies against HSP90 (heat shock protein 90) (1:500, ab203085, ABCAM), HSP60 (heat shock protein 60) (1:500, ab190828, ABCAM), CLPP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) (1:500, Cat #PA5-52722, Invitrogen) and HO-1 (heme oxygenase 1) (1:1000, ab52947, ABCAM), overnight at 4 ℃. The next day, membranes were washed three times in PBS (Phosphate buffered saline) for 5 min and incubated with anti-rabbit IRDye700CW secondary antibodies (1:5000, Licor) in PBS/0.5% Tween-20 for 1 h at room temperature. All the antibodies were diluted in Odyssey Blocking Buffer. The obtained blots were visualized by Odyssey Infrared Imaging Scanner (Licor, Milan, Italy). Densitometric analysis was used for protein levels quantification, normalizing data to protein levels of β-actin (1:2000, ab8229, ABCAM).
6
Western Blot Analysis of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain slices were collected and lysed in the radioimmunoprecipitation assay (RIPA) buffer [50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate] with a protease inhibitor. Equal amounts of proteins, measured by the BCA method, were loaded on 15% Bris-Tris NuPAGE, electrophoresed, and transferred into 0.22 μm nitrocellulose membranes. After blocking with 5% BSA in TBST for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-SNPH (ab69992, Abcam, USA), anti-Miro1 (ab188029, Abcam, USA), anti-Trak1 (ab28751, Abcam, USA), anti-HSP60 (ab190828, Abcam, USA), and anti-β-actin (ab115777, Abcam, USA) at a dilution of 1:1,000 (Tris-buffered saline with 0.1% Tween® 20 detergent).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!