The proteins were extracted using lysis buffer [10 mM Tris-HCl (pH 7.5), 1 mM ethylene-diaminetetraacetic acid, 10% glycerol, 0.5% NP-40 detergent, 400 mM NaCl, 4 µg/ml of aprotinin, phenylmethylsulfonyl fluoride and dithiothreitol]. The BCA protein assay was used for the protein determination. Total protein (10 µg loaded per lane) was separated using SDS-PAGE (on a 10% polyacrylamide gel), at 300 mA for 90 min and transferred on a nitrocellulose membrane (Invitrogen; Thermo Fisher Scientific, Inc.). Blocking of the membrane was performed using 4% skimmed milk for 60 min at room temperature. The protein expression levels of CPA4, E-cadherin and β-actin were assessed using western blot analysis. These proteins were detected using specific antibodies to CPA4 (1:200; HPA021030; Sigma-Aldrich; Merck KGaA), E-cadherin (1:1,000; M106; Takara Bio, Inc.), and β-actin (1:1,000; #3700; Cell Signaling Technology, Inc.). The membrane was incubated in primary antibodies for overnight at 4°C. β-actin served as a loading control. ECL anti-mouse or anti-rabbit IgG, peroxidase-linked whole antibody was used for secondary antibody (GE Healthcare Life Sciences, Little Chalfont, UK). The signals were detected using the ECL Western Blotting Detection System and an Image Quant LAS 4000 machine (GE Healthcare Life Sciences).
Hpa021030
Manufactured by Merck Group
HPA021030 is a laboratory equipment product from Merck Group. It is a general-purpose instrument designed for various laboratory applications. The core function of HPA021030 is to perform fundamental tasks required in scientific research and testing environments. No further details or interpretation about the intended use of this product are available.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Takara Bio (1 mentions)
Ecl western blotting detection system, by GE Healthcare (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Imagequant las 4000 machine, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Ab52492, by Abcam (1 mentions)
2 protocols using hpa021030
1
Protein Extraction and Western Blot Analysis
2
Immunohistochemical Analysis of Breast Cancer
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The commercial tissue array containing 140 cases of breast cancer patients were constructed by Shanghai Biochip Co. Ltd. as described. For all the specimens, clinicopathological information (Age, Gender, Grade, TNM stage, and follow-up data) was available. The expression of CPA4, p53 and ALDH1A1 in the tissues were evaluated by immunohistochemical staining with specific antibodies. According to UltrasensitiveTM S-P immunohistochemistry kit protocol (Maixin Biotechnology Development Co., Ltd.), standard Avidin-biotin complex peroxidase immunohistochemical staining was performed. Briefly, after deparaffinizationin xylene and graded alcohols, heated antigen retrieval was done in citrate buffer (10mmol/L pH 6.0) by water-bath kettle heating for 30min. Endogenous peroxidase was blocked in 0.3% hydrogen peroxide for 10 min. Nonspecific binding was blocked by incubation in 10% normal animal serum for 10min. Sections were incubated at 4°C for 24 h with primary antibodies including polyclonal antibody against anti-CPA4 (HPA021030, Sigma-Aldrich), ALDH1A1 (Abcam, ab52492) and anti-p53 antibody (ab28, Abcam). Biotinylated secondary antibody and horseradish peroxidase-labeled avidin were subsequently used, and color was developed using the diaminobenzidine method.
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