Total protein (tissue samples and cells) was extracted with the RIPA buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%, SDS-PAGE, Sangon Biotech, Shanghai, China) was conducted to isolate total protein. Next, the isolated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Roche) and then blocked with tris buffered saline tween (TBST) buffer containing 5% skim milk. After washing with TBST, the membranes were incubated with primary antibodies at 4 °C overnight, including anti-Cleaved PARP (#9541, 1:1000), anti-B-cell lymphoma/leukaemia-2 (Bcl-2) (#4223, 1:1000), anti-Bcl-2-associated x (Bax) (#2772, 1:1000), anti-KIF2A (ab197988, 1:200, Abcam, Cambridge, MA, USA), and anti-β-actin (#4967, 1:1000). Subsequently, the membranes were incubated with goat anti-rabbit IgG (#7077, 1:2000). Protein bands were visualized with an ImmunoStar LD (Wako Pure Chemical, Osaka, Japan). β-actin was used as a loading control. All antibodies were purchased from Cell Signaling Technology (Santa Cruz, California, USA), except for KIF2A.
Ab197988
Manufactured by Abcam
Sourced in United Kingdom
Ab197988 is a lab equipment product. It is a device used for the detection and quantification of target analytes in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Pvdf membrane, by Roche (1 mentions)
Anti kif2a, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti β actin, by Abcam (1 mentions)
Ab4051, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
Mab374, by Merck Group (1 mentions)
Vectastain universal elite abc kit, by Vector Laboratories (1 mentions)
Ab18197, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protein extraction kit, by Keygen Biotech (1 mentions)
Diaminobenzidine, by Thermo Fisher Scientific (1 mentions)
7 protocols using ab197988
1
Protein Expression Analysis Protocol
2
Immunohistochemical Analysis of KIF2A in ESCC
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The human ESCC organizations used in this article are taken from patients who are self-hospital, and these are surgically cut tissues. The ESCC tissue was first deparaffinized, rehydrated in successively different concentrations of ethanol bath, placed in a pressure cooker and soaked in citrate buffer, and the antigen was recovered after approximately 30 minutes. KIF2A was then immunostained with a polyclonal antibody (ab197988, Abcam plc, Cambridge, UK) at a 1:200 dilution. Enzymatic detection of antibodies. The results were centrally reviewed and scored by two independent pathologists who were blinded. The scores were divided into four grades which are 0, 1, 2, 3 respectively. The use of human samples in this study was approved by the Ethics Committee of Xingtai people’s hospital, in accordance with the Declaration of Helsinki. The research involving human participants experiments had been approved by our hospital and our equivalent committee. The participants provided their written informed consents to participate in this study.
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China). The isolated total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The primary antibodies including anti-KIF2A (1: 500, ab197988, Abcam, Cambridge, UK), anti-Bax (1: 1000, ab32503, Abcam), anti-caspase-3 (1: 500, ab4051, Abcam), anti-Bcl-2 (1: 1000, ab59348, Abcam), anti-cyclinD1 (1: 200, ab16663, Abcam), and GAPDH (1: 1000, ab8245, Abcam) were used in this study. Secondary antibodies included goat-anti-rabbit IgG H&L (1: 2000, ab6721, Abcam) and goat anti-mouse IgG H&L (1: 2000, ab205719, Abcam). Finally, the protein signals were measured by the enhanced chemiluminescence kit.
4
Immunoblotting and Immunohistochemistry Protocols
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Membranes were immunoblotted with rabbit polyclonal anti-KIF2A antibodies (1:1000 dilution; ab197988; Abcam, Cambridge, UK) and monoclonal anti-GAPDH antibodies (1:20000 dilution; MAB374; EMD Millipore, Billerica, MA, USA). The procedures for western blotting were as described previously [16 (link),18 (link)].
Immunohistochemistry was performed with a VECTASTAIN Universal Elite ABC Kit (catalog no: PK-6200; Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s protocol. The characteristics of patients included in the tissue microarray (catalog no: LC813a; US Biomax, Inc., Derwood, MD, USA) are shown inTable 3 . The procedure for immunohistochemistry is described in our earlier reports [16 (link),21 (link),50 (link)].
Immunohistochemistry was performed with a VECTASTAIN Universal Elite ABC Kit (catalog no: PK-6200; Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s protocol. The characteristics of patients included in the tissue microarray (catalog no: LC813a; US Biomax, Inc., Derwood, MD, USA) are shown in
5
Antibody and Primer Sequences for KIF2A and GAPDH
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The antibodies used were as follows: rabbit anti-KIF2A (1 : 200 dilution for IHC; 1 : 1000 dilution for immunoblot, ab197988, Abcam, Cambridge, UK) and mouse anti-β-actin (1 : 2000 dilution, ab8227, Abcam, Cambridge, UK).
The quantitative PCR primer sequences of KIF2A are shown as follows: forward 5′-CCTGACCTTGTTCCTGATGAAG-3′ and reverse 5′-TGCTGAACCAACCACTCTATTATC-3′. The quantitative PCR primer sequences of GAPDH are as follows: 5′-CGACCACTTTGTCAAGCTCA-3′ and reverse 5′-GGTTGAGCACAGGGTACTTTATT-3′.
The shRNA plasmids (ready-to-package AAV) of KIF2A were bought from Addgene, and the targeted sequences of the KIF2A shRNA plasmids were shown as follows: sense, 5′-AATACATCAAGCAATGGTAACAT-3′.
The quantitative PCR primer sequences of KIF2A are shown as follows: forward 5′-CCTGACCTTGTTCCTGATGAAG-3′ and reverse 5′-TGCTGAACCAACCACTCTATTATC-3′. The quantitative PCR primer sequences of GAPDH are as follows: 5′-CGACCACTTTGTCAAGCTCA-3′ and reverse 5′-GGTTGAGCACAGGGTACTTTATT-3′.
The shRNA plasmids (ready-to-package AAV) of KIF2A were bought from Addgene, and the targeted sequences of the KIF2A shRNA plasmids were shown as follows: sense, 5′-AATACATCAAGCAATGGTAACAT-3′.
6
Protein Extraction and Western Blot Analysis
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We used a protein extraction kit (KeyGEN Biotech, Nanjing, China). The concentration of the protein was measured using a Bio-Rad Protein Assay Kit (KeyGEN Biotech). The protein sample was subjected to electrophoresis and then electrotransferred to a PVDF membrane (Millipore, USA). After completion, the cells were blocked with 5% skim milk powder in TBST buffer for 1 hour. Then incubate with the corresponding antibody and overnight at 4 °C. The next day, it was incubated with the corresponding secondary antibody for 1 hour at room temperature. Then check the strip condition. This study used β-actin as an internal reference. In addition, the antibody information used herein is as follows: rabbit anti-KIF2A antibody (1:1, 0000 dilution, ab197988, Abcam plc, Cambridge, UK), rabbit anti-β-actin (1:1000 dilution, ab8227, Abcam plc, UK) Cambridge), rabbit anti-Ki67 (1:1000 dilution, ab16667, Abcam plc, Cambridge, UK) and rabbit anti-proliferating cell nuclear antigen (PCNA) (1:500 dilution, ab18197, Abcam plc, Cambridge, UK).
7
Semiquantitative Analysis of KIF2A Expression
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The FFPE specimens were processed by immunohistochemical staining for semiquantitative analysis of KIF2A expression. The IHC staining was implemented according to the standard procedures as described in a previous study.14 The following antibodies were respectively applied as primary and secondary antibodies: Rabbit polyclonal to KIF2A at a dilution ratio of 1/300 (ab197988, Abcam), and Goat Anti‐Rabbit IgG H&L (HRP) at a dilution ratio of 1/50,000 (ab6721, Abcam). Diaminobenzidine (Invitrogen) was used as chromogenic reagent and hematoxylin (Sigma‐Aldric) was applied for counterstaining. The IHC staining results were assessed by two pathologists independently. The staining intensity and the percentage of positive cells were respectively scored from 0 to 3 and from 0 to 4, referring to a previously published methodology.18 An IHC score was yielded by multiplying these two scores, and the mean score of two pathologists was used as final result in the analysis. The KIF2A expression was categorized as high (IHC score >3) and low (IHC score ≤3).18
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