Mycoplasma cell lysates were quantified using the Pierce ™ BCA Protein Assay Kit. Cell extracts were separated by electrophoresis using NuPage™ 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and proteins transferred onto nitrocellulose membranes using an iBlot™ dry system (Invitrogen). Membranes were blocked in a PBS buffer containing 0.1% Tween 20 supplemented with 5% skim milk (Sigma) or 3% BSA in the case of Strep-tag detection. For immunodetection, membranes were probed with polyclonal anti-CAT (abcam, 1:2,000), monoclonal anti-FLAG M2 (Sigma, 1:5,000), Strep-Tactin conjugated to horseradish peroxidase (IBA lifesciences, 1:10,000), DsRed polyclonal antibody (Takara, 1:2,000), anti-polyclonal anti-ssrAmk tag (1:2,000) or polyclonal antibodies specific to mycoplasma proteins (kind gift of Dr. Herrmann, Heidelberg University). These include anti-Lon (1:3,000) and anti-FtsH (1:3,000), which were also used as loading controls together with anti-CAT (abcam, 1:2,000) antibody. Anti-mouse IgG (1:10,000) or anti-rabbit IgG (1:5000) conjugated to horseradish peroxidase (Sigma) were used as a secondary antibody. Blots were developed with the Supersignal™ West Femto or West Pico Chemiluminescent Substrate detection Kit (Thermo Scientific) and signals detected in a LAS-3000 Imaging System (Fujifilm).
Anti cat
Manufactured by Abcam
Sourced in United Kingdom
Anti-CAT is a laboratory equipment product manufactured by Abcam. It is designed for the detection and quantification of Catalase (CAT) in various biological samples. The product provides a reliable and sensitive method for measuring Catalase activity.
Automatically generated - may contain errors
Lab products found in correlation
Anti sod1, by Abcam (3 mentions)
Las 3000 imaging system, by Fujifilm (2 mentions)
Skim milk, by Merck Group (2 mentions)
Anti sod2, by Abcam (2 mentions)
Strep tactin conjugated to horseradish peroxidase, by IBA Lifesciences (1 mentions)
Dsred polyclonal antibody, by Takara Bio (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Nupage 4 12 bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Anti p53, by Abcam (1 mentions)
Anti p21, by Abcam (1 mentions)
Anti gpx, by Abcam (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Instantblue, by Abcam (1 mentions)
Supersignal west pico chemiluminescent substrate detection kit, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Monoclonal anti flag m2, by Merck Group (1 mentions)
Anti cox 4, by Cell Signaling Technology (1 mentions)
7 protocols using anti cat
1
Quantitative Western Blot Analysis
2
Protein and Antioxidant Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells in each group were counted and lysed for protein quantification using a BCA kit (Pierce, USA). The amount of soluble protein was divided by the cell number as a marker of cellular hypertrophy (protein/cell). Western blotting analysis of cellular proteins was performed according to a previously described method [31] . For the analysis, total cellular proteins were isolated using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) 48 h after RAPA treatment. For the cell cycle regulatory protein analysis, the cells were lysed 72 h after the final UVB irradiation. Equal amounts of protein from each group were separated on 12% SDS-PAGE gels for 60 min and blotted onto polyvinyl difluoride membranes. The blots were incubated with anti-p53, anti-p21, anti-collagen I, anti-SOD1, anti-SOD2, anti-CAT, and anti-GPX antibodies (all from Abcam, Cambridge, UK) and then washed and incubated with a horseradish peroxidaseconjugated secondary antibody. The bands were visualized via chemiluminescence (Pierce, Rockford, USA).
3
Western Blot Analysis of Mycoplasma Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mycoplasma cell lysates were quantified using the Pierce™ BCA Protein Assay Kit, and 10 µg of cell extracts was subjected to electrophoresis through NuPAGE™ 4–12% Bis‐Tris pre‐cast polyacrylamide gels (Invitrogen). Proteins were then visualized after InstantBlue™ (Expedeon) Commassie staining or transferred onto nitrocellulose membranes using an iBlot™ dry blotting system (Invitrogen). When possible, membranes were cut at appropriate molecular weights and probed with different antibodies independently. For immunodetection, membranes were blocked with 5% skim milk (Sigma) in PBS containing 0.1% Tween 20 solution and probed with monoclonal anti‐FLAG M2 (Sigma) antibody (1:5,000), polyclonal anti‐Fluc (Invitrogen) antibody (1:4,000), or polyclonal antibodies specific to mycoplasma proteins (kind gift of Dr. Herrmann, Heidelberg University). These include anti‐Lon (1:3,000), anti‐FtsH (1:3,000), anti‐HMW1 (1:10,000), anti‐P65 (1:3,000), anti‐P30 (1:10,000), and anti‐RL‐7 (1:5,000). As a loading control, we used a polyclonal anti‐CAT (Abcam) antibody (1:2,000). Anti‐mouse IgG (1:10,000) or anti‐rabbit IgG (1:5, 000) conjugated to horseradish peroxidase (Sigma) was used as a secondary antibody. Blots were developed with the Supersignal™ West Pico Chemiluminescent Substrate detection Kit (Thermo Scientific) and signals detected in a LAS‐3000 Imaging System (Fujifilm).
4
Comprehensive Immunoblotting Analysis of Cellular Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The detailed procedure has been described24 (link). The following primary antibodies were used: anti-Fxn (GeneTex, Cat.#: GTX54036), anti-Cox4 (Cell Signaling Technology, Cat.#: 48445), total OXPHOS Rodent WB Antibody Cocktail (MitoSciences, Cat.#: MS604), anti-Sod1 (Abcam, Cat.#: ab16831), anti-Sod2 (Enzo Life Sciences, Cat.#: ADI-SOD-111-D), anti-Sod3 (R&D Systems, Cat.#: AF4817), anti-Cat (Abcam, Cat.#: ab15834), anti-β-Tublin (Cell Signaling Technology, Cat.#: 2146 S), anti-Mmp9 (Proteintech, Cat.#: 10375-2-AP), anti-Col1a2 (Proteintech, Cat.#: 14695-1-AP), anti-Irp1 (Santa Cruz Biotechnology, Cat.#: sc-166022), anti-Tfrc (Abcam, Cat.#: ab84036), anti-Ft (Abcam, Cat.#: ab75973), anti-Opa1 (Novus Biological, Cat.#: NBP2-34206), anti-4-HNE (Abcam, Cat.#: ab48506) and anti-Gapdh (Cell Signaling Technology, Cat.#: 14C10). The proteins were detected and quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
5
Antioxidant Enzyme Expression and Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression of MnSOD, CAT, GPx1 and β-actin was determined in RV lysates by immunoblot using specific antibodies (anti-Mn-SOD, Millipore, 06-984, 1:1000; anti-CAT, Abcam Laboratories, ab1877, 1:10,000; anti-GPx1, Abcam Laboratories, ab22604, 1:1000; and anti-β-actin, AC-15, Thermo Fisher scientific, 1:5000), as described elsewhere [34 ]. The signals obtained by immunoblot were scanned and quantified by densitometric analysis (Odyssey Imaging System, Li-Cor Biosciences, Lincoln, NE, USA).
The antioxidant enzymes activities in RV tissue homogenate were measured using the Superoxide Dismutase (SOD) Activity Assay Kit (K335-100, Biovision, quantified at 450 nm), the OxiSelect Catalase Activity Assay Kit (STA-341, Cell Biolabs Inc., quantified at 520 nm), and the Glutathione Peroxidase Assay Kit (703102, Cayman Chemical Company, Ann Arbor, MI, USA, quantified at 350 nm), according to the manufacturers’ guidelines. Total protein concentration was used for normalization purposes [26 (link)].
The antioxidant enzymes activities in RV tissue homogenate were measured using the Superoxide Dismutase (SOD) Activity Assay Kit (K335-100, Biovision, quantified at 450 nm), the OxiSelect Catalase Activity Assay Kit (STA-341, Cell Biolabs Inc., quantified at 520 nm), and the Glutathione Peroxidase Assay Kit (703102, Cayman Chemical Company, Ann Arbor, MI, USA, quantified at 350 nm), according to the manufacturers’ guidelines. Total protein concentration was used for normalization purposes [26 (link)].
6
Mitochondrial Function and Oxidative Stress Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of rat liver were homogenized as previously described [38 (link)]. Liver lysates containing 30 µg protein were loaded in each lane and were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membrane. Membranes were blocked and after probed with the following antibodies: anti-PGC1α (Millipore, Burlington, MA, USA), anti-NRF1 (Abcam, Cambridge, UK), anti-TFAM (Abcam), anti-TOM20 (Cell signaling), anti-GPX4 (Abcam), anti-PRDX3 (Abcam), anti-SOD2/MnSOD (Abcam), anti-CAT (Abcam), anti-DRP1 (Abcam), anti-OPA1 (Abcam), anti-MNF2 (Abcam), anti-PAMPK Thr172 (Cell signaling, Danvers, MA, USA), anti-AMPK (Cell signaling), anti-PULK Ser555 (Cell signaling), anti-ULK1 (Cell signaling), anti-AMBRA1 (Cell signaling), anti-PINK1 (Cell signaling ) anti-PARKIN (Cell signaling), anti-LC3B (Novus biologicals), anti-POLγ (Novus biologicals, Bio-Techne SRL, Milano, Italy), anti-Total OXPHOS complexes cocktail (Abcam), and anti-βACTIN (Sigma Aldrich, St. Louis, MO, USA). As secondary antibodies, peroxidase anti-rabbit IgG (Vector Laboratories Burlingame, CA, USA) and peroxidase anti-mouse IgG (Vector Laboratories) were used. Horseradish peroxidase-conjugated secondary antibodies were detected with enhanced chemiluminescence.
7
Evaluation of Immunomodulatory and Antioxidant Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPMI-1640 medium, fetal bovine serum (FBS), benzylpenicillin, and streptomycin were offered by Gibco (Grand Island, NY, USA). Dimethyl sulfoxide, ConA, trypan blue, and 5-diphenyl-tetrazolium bromide and 3-(4,5-dimethylthiazol-2-yl)-2 (MTT) were provided by Sigma-Aldrich Co., (St Louis, MO, USA). YAC-1 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cyclophosphamide was provided by Shengdi Pharmaceutical Co., Ltd. (Jiangsu, China). Levamisole hydrochloride tablets were obtained from Renhetang Pharmaceutical Co., Ltd. (Shandong, China). The antibodies for the T cell subpopulations assay were provided by BioLegend (San Diego, CA, USA), including fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4, allophycocyanin (APC)-conjugated anti-mouse CD8a, and phycoerythrin (PE)-conjugated anti-mouse CD25. Cytokines, including IL-1β, IL-4, IL-6, IFN–γ, and TNF-α, were provided by Invitrogen Co., Ltd. (Carlsbad, CA, USA). In this study, anti-Nrf2, anti-HO-1, anti-NQO1, anti-SOD1, anti-SOD2, and anti-CAT antibodies were provided by Abcam (Cambridge, MA, USA).
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!