Protein concentration was measured using a BCA Protein Assay Kit following the manufacturer’s protocol (Thermo Fisher Scientific). Proteins (25 μg/sample) were separated on a 4–12% SDS-PAGE gel and transferred onto nitrocellulose membrane blots using semi-dry blotting system (Bio-Rad) following the manufacturer’s protocol. To ensure the equal protein loading in each lane, the blots were stained Ponceau S (Sigma) and imaged on a ChemiDoc™ imaging system (Bio-Rad). Blots were then incubated with primary monoclonal/polyclonal antibodies including CFD (R&D systems, AF1824, 1:2500), GPX3 (R&D systems AF4199, 1:200), CST3 (Abcam ab133495, 1:13000), PON1 (Abcam, ab92466, 1:5000), MRC1 (Abcam ab195193, 1:1000) and COMP (Abcam, ab74524, 1 :200), followed by respective HRP-conjugated secondary antibodies. Blots were imaged using a Li-Cor Odyssey Blot imager (LI-COR Biosciences). Quantitation of signal intensity of the bands in Western blots was performed using Image lab software version 5.0 (Bio-Rad) and Image Studio Lite version 5.2 (LI-COR Biosciences).
Ab92466
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab92466 is a laboratory equipment product. It serves a core function for scientific research and analysis. No further details are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging system, by Bio-Rad (1 mentions)
Image studio lite version 5, by LI COR (1 mentions)
Image lab software version 5, by Bio-Rad (1 mentions)
Af1824, by R&D Systems (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Odyssey blot imager, by LI COR (1 mentions)
Ab133495, by Abcam (1 mentions)
Ab74524, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Semi dry blotting system, by Bio-Rad (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Protein g agarose slurry, by Roche (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Goat anti rabbit hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab52492, by Abcam (1 mentions)
Ab51037, by Abcam (1 mentions)
Ab223582, by Abcam (1 mentions)
Vinculin, by Abcam (1 mentions)
4 protocols using ab92466
1
Quantitative Western Blot Analysis
2
Immunoprecipitation of Serum Proteins
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Serum was diluted with lysis buffer (Beyotime, Shanghai, China). In this procedure, 20 µl of protein G agarose slurry (Roche, Basel, Switzerland) was added to the diluted serum at 4°C for 3–4 h incubation to remove IgG interference. After high speed centrifugation, 30 µl of protein G agarose slurry (Roche) and anti-PON1 antibody (ab92466, Abcam, UK) were sequentially added to the IgG-depleted serum for overnight incubation. Then the beads were washed with the same lysis buffer for five times. IP samples were handled with SDS-PAGE for in-gel digestion and glycopeptide enrichment.
3
Western Blot Protein Expression Analysis
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Protein expression was measured using western blot analysis, as described previously [41 (link)]. Briefly, proteins were separated by electrophoresis (SDS/PAGE) and transferred using a semi‐dry transfer system (Bio‐Rad, Hercules, CA, USA). This was followed by overnight incubation with listed primary antibodies at 4 °C, and subsequently with secondary antibodies for 1 h at RT. Primary antibodies against ALDH1A1 (ab52492; RRID:AB_867566), CD44 (ab51037; RRID:AB_868936), EpCAM (ab223582; RRID:AB_2762366), and PON1 (ab92466; RRID:AB_10562283) were purchased from Abcam while Vinculin (#13901; RRID:AB_2728768) and GAPDH (#5174; RRID:AB_10622025) from Cell Signaling Technology (Danvers, MA, USA). HRP‐conjugated Goat anti‐Rabbit secondary antibody from Bio‐Rad (1706515; RRID:AB_11125142) was used.
4
Immunohistochemical Analysis of PON1 in KIRC
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Tissue sample of KIRC fixed with formalin were used for Immunohistochemistry staining.
PON1 antibodies for immunohistochemical staining were obtained from Abcam (ab92466). We compared the staining between renal carcinoma and normal tissue under the microscope. The final scores depended on two kinds of factors: positive cells score and staining intensity score. Firstly, positive cell score: 0-5% is 0, 6-25% is 1, 26-50% is 2, 51-75% is 3 and greater than 75% is 4.
Besides, score of staining intensity: 0 for no staining, 1 for a slightly yellow background, 2 for a yellowish background and 3 for a brown background. Samples were collected from kidney cancer patients at the Affiliated Hospital of Nantong University. The ethical review was passed by the Affiliated Hospital of Nantong University. We performed immunohistochemical tests on 10 renal cancers tissue and 10 paracancerous tissues.
PON1 antibodies for immunohistochemical staining were obtained from Abcam (ab92466). We compared the staining between renal carcinoma and normal tissue under the microscope. The final scores depended on two kinds of factors: positive cells score and staining intensity score. Firstly, positive cell score: 0-5% is 0, 6-25% is 1, 26-50% is 2, 51-75% is 3 and greater than 75% is 4.
Besides, score of staining intensity: 0 for no staining, 1 for a slightly yellow background, 2 for a yellowish background and 3 for a brown background. Samples were collected from kidney cancer patients at the Affiliated Hospital of Nantong University. The ethical review was passed by the Affiliated Hospital of Nantong University. We performed immunohistochemical tests on 10 renal cancers tissue and 10 paracancerous tissues.
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