Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Total protein was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Subsequently, 15 µg protein/lane was separated using 8–10% SDS-PAGE and transferred onto a PVDF membrane, which was subsequently blocked with 5% skimmed milk diluted with TBS-1% Tween (TBST) for 2 h at room temperature. The PVDF membrane was then incubated overnight at 4°C with polyclonal antibodies for MMP-2 (cat. no. ab215986; 1:1,000; Abcam) and MMP-9 (cat. no. ab219372; 1:1,000; Abcam), with GAPDH antibody (cat. no. ab8245; 1:10,000; Abcam) serving as an internal control. The membrane was further incubated with horseradish peroxidase-conjugated anti-rabbit (cat. no. sc-2030) or anti-mouse (cat. no. sc-2005) immunoglobulin-G secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc.) diluted with TBST for 1 h at room temperature. The resulting protein signals were detected using an enhanced chemiluminescence reaction system (EMD Millipore) and quantified using ImageJ software (version 1.8.0; National Institutes of Health).
Ab215986
Manufactured by Abcam
Sourced in United States
Ab215986 is a laboratory equipment product offered by Abcam. It serves as a core function without further extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ab219372, by Abcam (4 mentions)
Ab205718, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab181602, by Abcam (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Ab93689, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab32572, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Gapdh antibody, by Abcam (1 mentions)
Bicinchoninic acid assay kit, by Beyotime (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab18203, by Abcam (1 mentions)
Ab9106, by Abcam (1 mentions)
Ab93926, by Abcam (1 mentions)
Ab75814, by Abcam (1 mentions)
Ab49314, by Abcam (1 mentions)
7 protocols using ab215986
1
Quantifying Cellular Protein Levels
2
Western Blot Analysis of EMT Markers
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The cell extracts were lysed with ice-cold lysis buffer. The protein concentration was calculated using BCA Protein Assay kit (23,227, Pierce) and equal amount of proteins were separated by SDS-PAGE, as described previously [17 (link)]. Then, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane which was blocked for 1 h with 5% nonfat milk and incubated with primary antibodies against Matrix metalloproteinase (MMP)2 (1:1000, 74kD, ab215986, Abcam), MMP9 (1:1000, 78kD, ab219372, Abcam), E-cadherin (1:10,000, 97kD, ab40772, Abcam), N-cadherin (1:1000, 130kD, ab18203, Abcam) and GAPDH (1:10,000, 36kD, ab181602, Abcam) at 4°C overnight, followed by the incubation with secondary antibody goat anti-rabbit IgG H&L (1:1000, ab205718, Abcam) for 1 h. Then, the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., MD, USA) was used to analyze proteins expressions. GAPDH was used as an internal control.
3
Western Blot Analysis of Protein Expression
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The extracted total protein was subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Following blockade, membranes were treated with primary antibodies overnight and with secondary antibody for 2 h in the dark. Primary antibodies against GLI1 (ab49314), SOX2 (ab93689), MYC (ab9106), VEGF (ab32152), β-catenin (ab32572), MMP2 (ab215986), p70S6K2 (ab184551), GSK3β (ab93926), p-GSK3β (Y216) (ab75745), p-GSK3β (S9) (ab75814), Cyr61 (ab24448), and GAPDH (ab8245), were all from Abcam (Cambridge, MA, USA). All samples were assayed in triplicate.
4
Protein Expression Analysis in Stem Cells
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Total cellular proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific), separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked using fat-free milk and incubated with specific antibodies at 4 °C overnight. The primary antibodies were all purchased from Abcam (Cambridge, MA, USA), including anti-Bax (ab32503), anti-Bcl-2 (ab185002), anti-MMP2 (ab215986), anti-CD44 (ab216647), anti-SOX2 (ab97959), anti-Oct4 (ab93689), anti-Nanog (ab218524), anti-β-catenin (ab32572), anti-cyclin D1 (ab1663), anti-c-myc (ab32072), and anti-GAPDH (ab8245). Membranes were then cultivated with secondary antibody (ab205718; Abcam) and detected by chemiluminescence system (Bio-Rad).
5
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using the RIP assay strong lysis buffer (Beyotime, Shanghai, China). Protein concentrations were quantified using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, United States). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was then performed to separate the proteins, which were then transferred onto a polyvinylidene fluoride membrane. Then, the membranes were incubated with primary antibodies (BLC2, 1:1,000, ab32124; MMP2, 1:2,000, ab215986; MMP9, 1:1,000, ab219372; caspase-3, 1:1,500, ab32351; caspase-9, 1:500, ab2324; and BAX, 1:1,000, ab199677; all purchased from Abcam, Cambridge, MA, United States) overnight at 4°C. Afterward, the membranes were washed and incubated with a secondary antibody (anti-rabbit IgG, HRP-linked Antibody, #7074, 1:5,000; Cell Signaling Technology, Beverly, MA, United States) at room temperature for 2 h. Finally, the bands on the membranes were visualized using enhanced chemiluminescence (Pierce, Rockford, IL, United States), and signals were exposed to X-ray. All films were scanned and densitometric analysis was performed three times using Image Pro-Plus 6.0 software (Media Cybernetics, Silver Spring, MD, United States). The relative protein abundance was expressed as the ratio of the target protein densitometric value to the GAPDH densitometric value.
6
Protein Expression Analysis of NSCLC Cells
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After relevant treatment, NSCLC cell lysate was prepared. The cell lysate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gel and blotted to the polyvinylidene fluoride (PVDF) membrane. After blocking, the membrane was then incubated with antiproliferating cell nuclear antigen (anti‐PCNA; ab92552; Abcam, Cambridge, MA, USA), anti‐Cyclin D1 (ab16663; Abcam), anti‐matrix metallopeptidase 2 (MMP2; ab215986; Abcam), anti‐MMP9 (ab219372; Abcam), anti‐NOVA2 (ab51004; Abcam) or anti‐GAPDH (ab181602; Abcam) followed by incubation with the secondary antibody (ab205718; Abcam). The protein signal was detected using the enhanced chemiluminescent (ECL) system (Beyotime, Shanghai, China).
7
Protein Expression Analysis in Cervical Cancer
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Total protein was obtained from transfected SiHa or CaSki cells which were lysed by radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Jiangsu, China). Subsequently, protein was quantified by using the BCA assay kit (Beyotime). The dodecyl sulfate, sodium salt (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed on proteins from lysates which were then moved to polyvinylidene fluoride membranes (PVDF; Millipore, Bedford, MA, USA). Membranes were cultured with primary antibodies overnight at 4 °C, and antibodies used were as follows: anti-Bcl-2 (ab32124, Abcam, Cambridge, USA), anti-Bax (ab32503, Abcam), anti-MMP2 (ab215986, Abcam), anti-MMP9 (ab219372, Abcam), anti-PBX1 (ab97994, Abcam) and anti-GAPDH (ab97994, Abcam). Then, secondary antibodies were added for cultivation for 1 h in dark room. Finally, the proteins were detected by an Enhanced Chemiluminescence Detection Kit (Millipore, Bedford, MA, USA).
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