The collected cells were lysed in PMSF buffer. The protein concentration was determined by using a BCA protein assay kit (Beyotime, China). Protein was separated by SDS-PAGE electrophoresis and transferred to membranes; membranes were subsequently blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 2 h. Next, the membranes were incubated with anti-LMX1A (ab106629; Abcam, Cambridge, MA, USA), anti-β-catenin (ab6302; Abcam), anti-TCF4 (ab217668; Abcam), and anti-β-actin (Abcam) overnight at 4℃. After washing, the membrane was then probed with specific secondary antibodies (Abcam) at room temperature for 2 h, and ECL visualization was performed. The protein bands were visualized and analyzed using Versa Doc™ imaging system.
Specific secondary antibody
Manufactured by Abcam
Sourced in United States
Specific secondary antibodies are laboratory reagents used to detect and visualize target proteins in various experimental techniques, such as Western blotting, immunohistochemistry, and ELISA. These antibodies are designed to bind to the Fc region of primary antibodies, enabling the detection and localization of the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Abcam (2 mentions)
Ab217668, by Abcam (1 mentions)
Ab106629, by Abcam (1 mentions)
Ab6302, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Ecl reagent, by Meilun (1 mentions)
Blocking buffer, by LI COR (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Lamin b1, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Specific primary antibody, by Abcam (1 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Ecl solution, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Imageexpress, by Molecular Devices (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
Ripa lysis buffer, by Boster Bio (1 mentions)
8 protocols using specific secondary antibody
1
Western Blot Analysis of LMX1A, β-catenin, and TCF4
2
Western Blot Protein Detection Protocol
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The methods have been described previously with slight modifications [26 (link),28 (link)]. Briefly, whole cell lysates were prepared in cell lysis buffer and clarified by centrifugation. Protein samples of 50 μg were separated on 8%–12% denaturing SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes by electroblotting. Membranes were blocked with blocking buffer (LI-COR, Lincoln, NE, USA) for 30 min at room temperature and then incubated with primary antibodies overnight at 4°C. Blots were incubated with specific secondary antibodies (Abcam, Cambridge, UK) at room temperature for 1 h the next day. The signals were detected by ECL reagents (Meilun Biological Technology, Dalian, China). β-actin (1:3000, Cell Signaling Technology, Danvers, MA, USA) and Lamin B1 (1:1000, Cell Signaling Technology, Danvers, MA, USA) were equivalent loading controls.
3
Protein Profiling of Cultured CRC Cells
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Proteins from cultured CRC cells were extracted by utilizing the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, P.R. China) according to standard protocol. DC Protein Assay Kit (Bio-Rad, P.R. China) was utilized to quantify the protein concentrations. Equal protein from each sample was loaded into SDS-polyacrylamide gels and separated through electrophoresis. Later proteins in the gels were transferred to polyvinylidene fluoride (PVDF) membranes. 3% bovine serum albumin (BSA) was used to block the membranes for 60 min at room temperature and then specific primary antibodies (Abcam, USA) were added to incubate at 4°C overnight. The antibodies were discarded and TBST was utilized to wash the membranes 3 times before incubation with specific secondary antibodies (1:20,000, Abcam) for 1 h at room temperature. Protein band intensities were detected by using the ECL kit (Bio-Rad). Primary antibodies used in the study were: anti-E-cadherin antibody (1:10,000), anti-N-cadherin antibody (1:1,000), anti-Vimentin antibody (1:2,000), anti-Snail (1:1,000), anti-HMGA1 antibody (1:10,000), anti-β-catenin antibody (1:5,000), anti-c-Myc (1:1,000), anti-Cyclin D1 antibody (1:25,000), and anti-GAPDH (1:2,500). The films were scanned, and the intensities were quantified using the ImageJ.
4
Western Blot Analysis of Cell Lysates
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bEnd.3 cells were washed with PBS and collected. The cell pellets were lysed with cold RIPA 20 min at 4°C to produce whole-cell extracts. Protein (25 μg) in cells was separated on a 12% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. After blocking with skim milk prepared in tris-buffered saline—Tween (TBST) (20 nM tris, pH 7.2, 150 mM NaCl, 0.1% Tween 20) for 1 hour and 30 min at room temperature, immunoblots were incubated for 14 hours at 4°C with primary antibodies that detect cleaved poly ADP-ribose polymerase (PARP) (1 : 1000, Abcam, Cambridge, MA, USA), claudin 5 (CLD5) (1 : 1000, Cell Signaling, Danvers, MA, USA), or β-actin (1 : 1000; Millipore, Billerica, MA, USA). Blots were then incubated with specific secondary antibodies (Abcam, Cambridge, MA, USA) for 2 hours at room temperature. Blots were visualized by ECL solution (Millipore, Billerica, MA, USA).
5
DNMT3A Protein Immunohistochemistry
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After deparaffinization and rehydration by xylene, absolute ethanol, and 70% alcohol, slides were drained, citrate buffer was used for antigen retrieval, and PBS was used for washing, followed by 3% H2O2 and 5% goat treatment. Anti- DNMT3A (1:200, LSBio, USA) and specific secondary antibodies (1:500, Abcam, USA) were utilized to incubate the slides under the condition of 4°C overnight and room temperature for 2 h, respectively. DAB staining reagent (Beyotime, China) was utilized to stain the slides and then photographed under microscopy.
6
Quantifying Carotid Plaque Composition
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Plaques obtained from human carotid endarterectomy were fixed in formalin, embedded in paraffin and sectioned at 5μm with a rotary microtome. Sections were incubated with anti-CD68, α-actin and SLAMF7 primary antibodies (Abcam Biotechnology company, USA) at room temperature and subsequently exposed to specific secondary antibodies (Sigma-Aldrich, USA). Whole-slide montage images were taken by the automatic microscope ImageExpress (Molecular Device, USA). Images were digitalized and the relative staining areas were quantified by Image J. A histology score (H score) was calculated by multiplying the fraction of positively stained cells (percentage) by staining intensity (0, 1+, 2+, or 3+) [31 (link)]. Intensity of immunoreactivity was scored (0 and 1+ indicates negative; 2+, indeterminate; and 3+, positive for overexpression), and the percentage of staining positive was visually estimated by certified pathologist at the Pathology Department of Liaocheng Hospital.
7
Protein Expression Analysis in KMRC Cells
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A RIPA lysis buffer (BOSTER, Wuhan, China) was used to extract total proteins from KMRC-1 or KMRC-3 cells. The proteins were then transferred onto PVDF membranes after being electrophoretically segregated on a SDS polyacrylamide gel (10%). 5% nonfat milk was used to block the membranes for 1 h at room temperature, and incubated with corresponding primary antibodies FN1 (Abcam, Cambridge, UK; cat.no. ab2413; 1:1000) and GAPDH (Abcam; cat.no. 9485; 1:1000) at 4°C overnight, and further incubated with specific secondary antibody (Abcam; cat.no. ab6721; 1:5000) at 25°C for 30 min. Internal control was performed using GAPDH. The protein bands were visualized using ECL kit (Tanon, Shanghai, China) on a Tanon3500 gel imaging system (Tanon), and quantified using Alpha Innotech software.
8
Quantifying Protein Expression in HeLa and CaSki Cells
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HeLa and CaSki cells were dissolved in RIPA lysis buffer (BOSTER, Wuhan, China) for total protein extraction. Thereafter, their concentrations were quantified using the Rapid Gold BCA Kit (Pierce, Rockford, IL, USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) was used to electrophorese a total of 30 µg of protein before they were moved to polyvinylidene fluoride membranes. The membranes were then sealed by being treated for 1 h with 5% nonfat milk at room temperature. Afterward, primary antibodies, namely MMP2 (Abcam, Cambridge, UK; 1:1,000) and GAPDH (Abcam; 1:1,000), were added and maintained overnight at 4°C. Thereafter, they were further incubated for 30 min at 25°C with a specific secondary antibody (Abcam; 1:3,000). GAPDH was used as the internal control. An enhanced chemiluminescence detection kit (Tanon, Shanghai, China) and Gel-Pro Analyzer 4.0 (Media Cybernetics, Silver Spring, MD, USA) were used to visualize and analyze the bands.
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