API (A3145) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS), antibiotics, molecular weight standards, trypsin-EDTA, and all medium additives were obtained from Life Technologies (Gaithersburg, MD). The CellTiter 96 AQueous One Solution Proliferation Assay System was purchased from Promega (Madison, WI). An enhanced chemiluminescence (ECL) kit was purchased from Amersham (Arlington Heights, IL). Antibodies specific for fibronectin (ab2413) and SPOCK1 (ab229935) were obtained from Abcam (Cambridge, MA). Antibodies specific for cleaved-PARP (#9541), phospho-Ak (ser473, #9271), Akt (#4691), Snail (#3895), Slug (#9585), Twist (#46702), and E-cadherin (#3195) were obtained from Cell Signaling Technology (Danvers, MA). Antibodies specific for vimentin (550513) and N-cadherin (610920) were purchased from BD Biosciences (San Jose, CA). Antibodies specific for cyclin D1 (sc-8396), cyclin E (sc-377,100), β-actin (sc-47,778), and goat anti-rabbit (sc-2004) and anti-mouse (sc-2005) immunoglobulin G (IgG) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene fluoride (PVDF) membranes for Western blotting were purchased from Bio-Rad (Hercules, CA). Unless otherwise specified, other chemicals used in this study were purchased from Sigma Chemical (St. Louis, MO).
Ab229935
Manufactured by Abcam
Sourced in United States
Ab229935 is a recombinant monoclonal antibody that recognizes the human CD3 epsilon antigen. This antibody is suitable for use in various immunoassays, including ELISA and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
E cadherin 3195, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Sc 8396, by Santa Cruz Biotechnology (1 mentions)
Molecular weight standards, by Thermo Fisher Scientific (1 mentions)
Api a3145, by Merck Group (1 mentions)
Polyvinylidene fluoride pvdf membranes for western blotting, by Bio-Rad (1 mentions)
Celltiter 96 aqueous one solution proliferation assay system, by Promega (1 mentions)
N cadherin 610920, by BD (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ecl substrate kit, by Cytiva (1 mentions)
Ab233611, by Abcam (1 mentions)
Ab254512, by Abcam (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Ab92547, by Abcam (1 mentions)
Ultravision quanto detection system hrp dab kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using ab229935
1
Comprehensive Materials and Reagents for Cell Assays
2
Western Blot Analysis of EMT Markers
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RIPA lysis buffer (Beyotime, Shanghai, China) was employed for lysing HCC cells to extract the total protein, and BCA Protein Assay (Thermo-Fisher Scientific, Waltham, MA, USA) was utilized for quantifying the protein in the samples. Subsequently, 20 μg protein in each group was separated using SDS-PAGE. Following the electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the membranes and primary antibodies (anti-SPOCK1: 1:2000, ab229935, Abcam, USA; anti-E-cadherin: 1:2000, ab233611, Abcam, USA; anti-Vimentin: 1:2000, ab92547, Abcam, USA; anti-N-cadherin: 1:2000, ab254512, Abcam, USA; anti-GAPDH: 1:5000, ab9485, Abcam, USA) were incubated overnight at 4°C, and then the membranes and secondary antibody (goat anti-rabbit IgG-HRP, 1:10000, ab6721; Abcam, USA) were incubated for 1 h at room temperature. Ultimately, enhanced chemiluminescence (ECL) substrate kit (Amersham Biosciences, Little Chalfont, UK) was employed to show the protein bands, and GAPDH acted as the internal reference.
3
Quantitative Gene and Protein Analysis
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RT-qPCR and Western Blot were conducted as previously described [21 (link)]. RT-qPCR was performed with the following primer pairs:
SLC6A14 forward 5′-GCTTGCTGGTTTGTCATCACTCC-3′ and reverse 5′-TACACCAGCCAAGAGCAACTCC-3′;
SPOCK1 forward, 5′-GTTCTACTGGCAAAAGCCTCGC-3′ and reverse 5′-AGGTTCCGCAACTCCTTGTCTG-3′;
GAPDH forward 5′-GGTGTGAACCATGAGAAGTATGA-3′ and reverse 5′-GAGTCCTTCCACGATACCAAAG-3
4
Immunohistochemistry of FORMALIN-FIXED Tissue
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Formalin-fixed paraffin-embedded specimens were used for immunohistochemistry as our previous study [17 (link)]. The tissue sections were deparaffinized in xylene and rehydrated using a graded ethanol series. To quench endogenous peroxidase activity, the sections were immersed in a 0.3% peroxidase-methanol solution for 30 min. For antigen retrieval, the sections were pretreated with citrate buffer for 15 min at 100 °C in a microwave oven. The materials and methods including immunohistochemistry and reagents referred to these research [18 , 19 (link), 20 (link)]. The sections were hybridized with a primary antibody against SLC6A14 (ab254786) and SPOCK1 (ab229935) at 4 °C overnight at a dilution of 1:1000 and were visualized using the UltraVision Quanto Detection System HRP DAB kit (Thermo Scientific, Shanghai, China) according to the manufacturer’s protocols. The stained sections were counterstained with hematoxylin, and photomicrographs were captured using an Olympus BX51 microscope. The following antibodies were purchased from Abcam: SLC6A14 (ab254786) and SPOCK1 (ab229935).
5
Protein Extraction and Western Blot Analysis of GBC
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Total protein of GBC tissues and cells was extracted using RIPA protein extraction reagent (Beyotime, Shanghai, China). Total protein were isolated by electrophoresis on 10% SDS-PAGE and transferred onto the PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked in TBST buffer with 5% non-fat milk for 1 h. Then, the membranes were incubated with primary antibodies: anti-cleaved caspase-3 (ab32042, 1:1,000, Abcam, Cambridge, MA, USA), anti-Bax (ab182733, 1:1000, Abcam), anti-Bcl-2 (ab32124, 1:1000, Abcam), anti-SPOCK1 (ab229935, 1:2000, Abcam), and anti-GAPDH (ab9485, 1:1000, Abcam). Subsequently, the membranes were incubated with secondary antibodies: goat antirabbit IgG (ab6721, 1:10000, Abcam). The immunoblot was visualized through the enhanced chemiluminescence solution (Beyotime).
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