Protein analyses were carried out according to a previously described method 29 (link). After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris-HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to nitrocellulose membranes. A rabbit polyclonal antibody against human HSP-70 purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA, dilution 1:500) was used to detect HSP-70. Cellular β-actin protein was immunodetected using a mouse monoclonal antibody against mouse β-actin (Sigma, dilution 1:2500) as the internal standard. The secondary antibodies used were horseradish peroxidase (HRP) anti-rabbit (Santa Cruz Biotechnology, dilution 1:2500) and HRP anti-mouse (Sigma, dilution 1:2500). After adding enhanced chemiluminescence substrates to react with these secondary antibodies according to the instruction of an enhanced chemiluminescence detection system of the Western Lightning Plus-ECL (Perkin Elmer), these protein bands were observed and quantified using a digital imaging system (UVtec, Cambridge, UK).
Horseradish peroxidase hrp anti rabbit
Manufactured by Santa Cruz Biotechnology
Horseradish peroxidase (HRP) anti-rabbit is a secondary antibody conjugate. It is used in immunodetection techniques to detect and visualize the presence of rabbit primary antibodies.
Automatically generated - may contain errors
2 protocols using horseradish peroxidase hrp anti rabbit
1
Western Blot Analysis of HSP-70
2
Extraction and Characterization of Entermorpha Prolifera Polysaccharides
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Celecoxib (Pfizer Inc., Manhattan, NY), Ketoprofen (Swiss Co., Ltd., Tainan,
Taiwan), Zoletil (Virbac., Grasse, Carros, France), and recombinant mouse IL-1β
(ACROBiosystems., Newark, DE) were purchased from commercial companies. p38
(MAPK) beta and AP1/JUN antibodies were purchased from Thermo Fisher Scientific
Inc. (Waltham, MA). Rabbit polyclonal COX-2 and TNF-α, horseradish peroxidase
(HRP)-anti-rabbit, or mouse IgG antibodies were purchased from Santa Cruz
Biotechnology, Inc. (Delaware, CA). Entermorpha prolifera was used to prepare
EPEs included hot water polysaccharide extract (HPE), basic polysaccharide
extract (BPE), and acidic polysaccharide extract (APE), provided by the Kinmen
Fisheries Research Institute, Taiwan. Pulverized EPEs were extracted at a
vehicle to raw material ratio of 50 mL/g via ultrasonic-assisted extraction
using a frequency of 40 kHz at 100 °C over a period of 1.5 h. Neutralized
water-soluble EPEs were precipitated by the addition of 95% EtOH (v/v) over a
period of 48 h, followed by protein depletion. Finally, the extracts were
concentrated using a rotary evaporator and powdered using a freeze-drying system
(Kingmech, FD 20L-6S, Taipei, Taiwan). The individual total polysaccharide yield
was determined via the phenol-sulphuric acid method using glucose as a standard
reference.
Taiwan), Zoletil (Virbac., Grasse, Carros, France), and recombinant mouse IL-1β
(ACROBiosystems., Newark, DE) were purchased from commercial companies. p38
(MAPK) beta and AP1/JUN antibodies were purchased from Thermo Fisher Scientific
Inc. (Waltham, MA). Rabbit polyclonal COX-2 and TNF-α, horseradish peroxidase
(HRP)-anti-rabbit, or mouse IgG antibodies were purchased from Santa Cruz
Biotechnology, Inc. (Delaware, CA). Entermorpha prolifera was used to prepare
EPEs included hot water polysaccharide extract (HPE), basic polysaccharide
extract (BPE), and acidic polysaccharide extract (APE), provided by the Kinmen
Fisheries Research Institute, Taiwan. Pulverized EPEs were extracted at a
vehicle to raw material ratio of 50 mL/g via ultrasonic-assisted extraction
using a frequency of 40 kHz at 100 °C over a period of 1.5 h. Neutralized
water-soluble EPEs were precipitated by the addition of 95% EtOH (v/v) over a
period of 48 h, followed by protein depletion. Finally, the extracts were
concentrated using a rotary evaporator and powdered using a freeze-drying system
(Kingmech, FD 20L-6S, Taipei, Taiwan). The individual total polysaccharide yield
was determined via the phenol-sulphuric acid method using glucose as a standard
reference.
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