Total protein was extracted from seven human glioma cell lines and one human microglia cell line (HMC3). 30 ug of protein was loaded onto 10% SDSPAGE and electrophoretically transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibody against CHI3L2 (1:1000 dilutions, rabbit polyclonal anti-CHI3L2, #22164, SAB, Maryland, USA). The membranes were then incubated with horseradish peroxidase-linked anti-rabbit antibody (at a 1:3000 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA). B-Actin was served as a loading control.
Horseradish peroxidase linked anti rabbit antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Horseradish peroxidase-linked anti-rabbit antibody is a laboratory reagent used for detection and visualization in various immunoassay techniques. It consists of an antibody directed against rabbit immunoglobulins, conjugated with the enzyme horseradish peroxidase. This conjugate can be used to detect the presence of rabbit-derived proteins or antibodies in experimental samples.
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Lab products found in correlation
Westernbright ecl kit, by Advansta (1 mentions)
Anti gfp, by Abcom (1 mentions)
Quick start bradford protein assay kit, by Bio-Rad (1 mentions)
Anti β actin, by Abcom (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Bca protein assay kit, by Abcam (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
4 protocols using horseradish peroxidase linked anti rabbit antibody
1
Quantifying CHI3L2 Protein Levels in Glioma Cell Lines
2
Quantitative Western Blot Analysis
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Proteins of the interest tissues (heart, skin, liver, muscle and testis) were isolated from transgenic mice that were confirmed by RT-PCR (non-transgenic pups as negative control). The protein concentration was determined using a Quick Start™ Bradford Protein Assay Kit (Bio-Rad, USA). Protein complexes were separated by SDS-PAGE, and transferred to nitrocellulose membranes (Hybond ECL, USA). Membranes were probed using the following primary antibodies: anti-β-actin (Abcom, 1: 1,000), anti-GFP (Abcom, 1: 1,000). Secondary antibodies were horseradish peroxidase-linked anti-rabbit antibody (Santa Cruz, 1: 2,000). Protein bands were visualized on a Bio-Rad Chemidoc XRS using a Western Bright ECL Kit (Advansta, Menlo Park, CA, USA).
3
Western Blot Analysis of Renal Proteins
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Protein expression was analyzed by western blot analysis. The primary antibodies used were as follows: rabbit polyclonal anti-α-SMA antibody (Sigma-Aldrich) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (BOSTER, Wuhan, China). The renal tissue was lysed in lysis regent and after 5 min centrifugation at 10,000 ×g, the supernatant was collected and protein levels were estimated by BCA protein assay kit (Beyotime). Protein (40 μg) was loaded equally in each well on 8% polyacrylamide gels for gel-electrophoresis and then electransferred to polyvinylidene difluoride membranes. After blocking the blots for 1 h with 5% skimmed milk, the gels were incubated separately for 16 h with rabbit polyclonal anti-α-SMA antibody (1 : 1000 dilution), mouse anti-GAPDH (1 : 1000 dilution), rabbit polyclonal anti-TGF-β1 antibody (1 : 1000 dilution), rabbit polyclonal anti-AT1 antibody (1 : 1000 dilution), and rabbit polyclonal anti-Renin antibody (1 : 1000 dilution). A secondary antibody was conjugated with horseradish peroxidase-linked anti-rabbit antibody (1 : 5000, Santa Cruz Biotechnology)
4
Western Blot Analysis of Renal PI3K/Akt Pathway
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Western blot techniques were employed to assess the protein expressions of PI3K, Akt and pAkt in renal tissue (homogenates). Protein levels were estimated by BCA protein assay kit from Biovision (USA). A 50-µg aliquot of protein was loaded equally in each well on 10% polyacrylamide gels and electrotransferred to polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, USA) at room temperature by blocking with a tris-buffered saline (TBS) solution, which contains tween 20 and 5% skimmed milk. The membrane was incubated at 4°C for 2 h with different primary antibodies like rabbit polyclonal anti-PI3K (1:1000; Santa Cruz Biotechnology, USA), anti-Akt and pAkt antibody (Ser 473-1:2000; Cell Signaling Technology, USA) or rabbit monoclonal anti-rat β-actin (1:500; Zhongshan Biotechnology, China), which served as internal control, and washed with TBS. A secondary antibody was conjugated with horseradish peroxidase-linked anti-rabbit antibody (1:2000 Santa Cruz Biotechnology) in TBS at room temperature for 1 h, and washed twice with TBS to remove unbound antibodies. The bounded antibodies were visualized using an enhanced chemiluminescence western blotting detection kit (INtRON Biotechnology Co., Ltd., Korea) and the protein expression (band) was quantified using the Image-Pro Plus software (Media Cybernetics, Inc., USA).
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