The cell lysates were collected, and 30 mg protein of each sample was separated using 10% SDS-PAGE. The protein was then electrotransferred onto Polyvinylidene Fluoride (PVDF) membrane (Millipore, Boston, MA, USA). The PVDF membrane was sealed in 10% skimmed milk. Then the membrane was incubated with primary antibodies, including anti-TNC (Abcam, ab108930, 1:1000 dilution), anti-GAPDH (Abcam, ab181602, 1:10,000 dilution), and anti-ERK (Abcam, ab184699, 1:1000 dilution)), anti-p-ERK (Abcam, ab201015, 1:1000 dilution), anti-MMP9 (Abcam, ab76003, 1:1000 dilution), anti-MMP2 (Abcam, ab92536, 1:1000 dilution), anti-E-cadherin (Abcam, ab1416, 1:1000 dilution), anti-N-cadherin (Abcam, ab18203, 1:1000 dilution), anti-vimentin (Abcam, ab92547, 1:1000 dilution), anti-snail (Abcam, ab216347, 1:1000 dilution), anti-clumping (Abcam, ab27568, 1:1000 dilution) and anti-TWIST1 (Abcam, ab50887, 1:1000 dilution) overnight. Then the membrane and horseradish peroxidase-conjugated secondary antibody (Abcam AB6721, 1:10,000 dilution) were incubated for 2 h at room temperature. Chemiluminescence detection reagent (Millipore, Boston, MA, USA) was used to visualize protein bands.
Ab27568
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab27568 is a laboratory product manufactured by Abcam. It serves as a core functional component for various research applications. The detailed specifications and intended use of this product are not available for a concise and unbiased presentation.
Automatically generated - may contain errors
Lab products found in correlation
Ab92547, by Abcam (8 mentions)
Ab50887, by Abcam (7 mentions)
Ab216347, by Abcam (6 mentions)
Ab53519, by Abcam (6 mentions)
Ab18203, by Abcam (5 mentions)
Ab40772, by Abcam (5 mentions)
Ab76011, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Ab6721, by Abcam (4 mentions)
Ab1416, by Abcam (4 mentions)
Ab231303, by Abcam (4 mentions)
Ab8245, by Abcam (4 mentions)
Ab76003, by Abcam (3 mentions)
Ab92536, by Abcam (3 mentions)
Ab82846, by Abcam (3 mentions)
Ab38898, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab181602, by Abcam (2 mentions)
Ab137321, by Abcam (2 mentions)
38 protocols using ab27568
1
Protein Expression Analysis Protocol
2
DCIS and Early Breast Cancer Profiles
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Patients with early‐stage breast cancer were identified from the London Breast Cancer Database on the basis of having either DCIS only (stage 0) or DCIS with an associated invasive component (stage I; ≤ 2 cm, and either pN0 or pN0mi). Cohort 1 consisted of 186 patients with low‐, intermediate‐ or high‐grade DCIS with no invasion or early invasion (stage 0 or stage I). Cohort 2 consisted of 118 patients with non‐high‐grade DCIS with the aforementioned characteristics, with either no invasion or early invasion (stage 0 or stage I). Clinicopathological variables for cohort 1 and cohort 2 patients entered into this study are listed in Table 1 . There was no overlap between patients in cohort 1 and cohort 2. The study was conducted under a protocol approved by the Western University Health Sciences Research Ethics Board and the Clinical Research Impact Committee of Lawson Health Research Institute. Immunohistochemical staining was conducted using the following antibodies: TBX3 (Abcam, Cambridge, MA, USA; ab99302; 1/200), SLUG (Abcam; ab27568; 1/750), and TWIST1 (Abcam; ab50887; 1/750). Details relating to immunohistochemistry protocol and quantification methods are located in the supplementary material, Supplementary materials and methods.
3
Immunoblotting Analysis of Epithelial-Mesenchymal Transition Markers in Breast Cancer
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Total proteins of BC tissues and cells were extracted by high‐speed centrifugation at 10 000 g/min for 10 min after lysis in RIPA buffer (Beyotime). Proteins (50 μg) were loaded into and isolated by 10% SDS‐PAGE. Isolated proteins were transferred onto nitrocellulose membrane and incubated with 5% low‐fat milk for non‐specific site blocking. Afterwards, membranes were probed with primary antibodies against YBX‐1 (Rabbit, 1:1000, ab12148, Abcam), Slug‐1 (Rabbit, 1:1000, ab27568, Abcam), Vimentin (Rabbit, 1:3000, ab137321, Abcam), N‐cadherin (Rabbit, 1:1000, ab18203, Abcam), E‐cadherin (Rabbit, 1:10 000, ab40772, Abcam), β‐catenin (Rabbit, 1:4000, ab6302, Abcam), c‐Jun (Rabbit, 1:1000, ab131497, Abcam), and β‐actin (Rabbit, 1:5000, ab179467, Abcam). After overnight incubation, membranes were incubated for 2 h with the indicated secondary antibodies. Bands were visualized using enhanced chemiluminescence reagent (EMD, Millipore).
4
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Radioimmunoprecipitation assay (RIPA) was utilized to extract total cellular protein content, after which a BCA assay (Beyotime, Shanghai, China) was used for protein quantification. Samples were then electrophoretically separated and transferred onto PVDF membranes (Millipore, MA, USA). Following blocking for 2 h with 5% nonfat milk, blots were examined with proper primary and secondary antibodies. The bands of protein were then discovered via enhanced chemiluminescence (ECL) and analyzed with ImageJ (NIH, MD, USA). Antibodies used in this study were as follows: E-cadherin (abcam, ab231303, 1 : 1000), N-cadherin (abcam, ab98952, 1 : 1000), Vimentin (abcam, ab92547, 1 : 1000), Slug (abcam, ab27568, 1 : 1000), Snail (abcam, ab216347, 1 : 1000), HRP Goat Anti-Rabbit IgG (H+L) (ABclonal, AS014, 1 : 4000), HRP Goat Anti-Mouse IgG (H+L) (ABclonal, AS003, 1 : 5000).
5
Protein Expression Analysis Protocol
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After transfection for 24 h, cells in the six-well plate were placed on ice. After extracting by RIPA lysate (with protease inhibitor), the concentrations of protein were determined by the BCA method. Then, proteins were heated at 95°C for 5 min before isolating by SDS–PAGE, and they were transferred onto a PVDF membrane subsequently. The membrane was then blocked for 1 h using 5% skim milk at an ambient temperature. Following that, the membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study were as follows: SLC12A8 (ab122152, Abcam), E-cadherin (ab40772, Abcam), β-catenin (ab227499, Abcam), vimentin (ab45939, Abcam), snail (ab82846, Abcam), and slug (ab27568, Abcam). After incubation, the membrane was taken out and washed using TBST (tris buffered saline with Tween) for 3 × 5 min. Subsequently, bands were finally examined by ECL (enhanced chemiluminescence) after incubating the membrane with the secondary antibody at an ambient temperature for 1 h. The gray values were scanned by Quantity One software, and the ratio of target protein/GAPDH was calculated.
6
Protein Extraction and Western Blot Analysis
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Proteins were collected from cells using the Whole Cell Lysis Assay Kit (KGP250, KeyGen, Nanjing, China). The protein concentration was determined using the bicinchoninic acid (BCA) method using the BCA Protein Quantitation Assay Kit (KeyGen, Nanjing, China). Protein was electrophoretically separated by 10% or 15% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA). The membranes were blocked for 1 hour with 5% bovine serum albumin (BSA) in TBS-T and incubated with specific primary antibodies overnight at 4° C followed by incubation with rabbit or mouse radish peroxidase-coupled secondary antibodies for 1 hour. Antibody binding was detected using the enhanced chemiluminescence reagent (Millipore, Billerica, MA). The antibodies used in this study were as follows: ALKBH5 (ab195377, Abcam), H3K27ac (#8173, Cell Signaling Technology [CST]), cyclin D1 (#2978, CST), cyclin E1 (ab33911, Abcam), c-Myc (#5605, CST), caspase-3 (19677-1-AP, Proteintech), BCL-2 (12789-1-AP, Proteintech), MMP2 (#87809, CST), MMP7 (#71031, CST), MMP9 (#13667, CST), E-cadherin (#3195, CST), N-cadherin (#13116, CST), vimentin (#5741, CST), β-catenin (#8480, CST), Snail (ab53519, Abcam), Slug (ab27568, Abcam), β-actin (ab8227, Abcam), and GAPDH (#8884, CST).
7
Western Blot Analysis of GBM Pathways
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Protein expressions of AEBP1, phosphorylated IκBα, IκBα, phosphorylated NF-κB p65, NF-κB p65, Cyclin D1 (CCND1), MYC Proto-Oncogene (c-Myc), Matrix Metallopeptidase 9 (MMP9), and Snail Family Transcriptional Repressor 2 (Slug) were detected by western blot. GBM cells were lysed with RIPA buffer containing protease inhibitors. Total proteins were separated by electrophoresis in 10% SDS-PAGE and subsequently transferred to PVDF membranes. The membranes were then incubated with primary antibodies overnight at 4°C, followed by an incubation with secondary antibodies for 1 h at room temperature. Immunoreactive bands were revealed by chemiluminescence, and relative expression of the target protein was normalized to that of GAPDH used as an internal reference. The primary antibodies used in this study were purchased from Abcam (USA): anti-AEBP1 (ab168355), anti-IκBα (ab7217), anti-p-IκBα (ab133462), anti-NF-κB p65 (ab16502), anti-p-NF-κB p65 (ab86299), anti-CCND1 (ab16663), anti-c-Myc (ab39688), anti-MMP9 (ab38898), anti-Slug (ab27568), and anti-GAPDH (ab8245).
8
Immunofluorescence Analysis of EMT Markers
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A549, H1299, SPCA-1, and PC9 cells were cultured on the Glass Bottom Cell Culture Dish for 24 h and then were fixed with 4% paraformaldehyde, permeabilized with 0.25% of Triton X-100 (Solarbio), and treated with 5% BSA. After being washed, the cells were probed with 1:100 diluted antibodies against GALNT6 (sc-100755, Santa Cruz Biotechnology), E-cadherin (13-1700, Invitrogen), N-cadherin (sc-393933, Santa Cruz Biotechnology), Slug (ab27568, Abcam), GRP78 (ab21685, Abcam) overnight at 4 °C. Subsequently, the cells were incubated with fluorophore-conjugated secondary antibodies (1:100, Proteintech) for 1 h and stained with DAPI (Sigma) for nuclei, followed by photoimaging under a confocal laser scanning microscope (Leica DM14000B).
9
Protein Expression Profiling of Cancer Markers
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The cell protein samples were extracted in 6-well plates using RIPA lysis buffer with protease inhibitor, then quantitated by BCA kit (Thermo Fisher Scientific), diluted in loading buffer to the same concentration, denatured at 95°C. Then, 20 μg proteins was subjected to SDS-PAGE for 2h and then transferred to PVDF membranes. After being blocked with skim milk, the membranes were co-cultured with primary antibodies against anti-KCNC4 (ab93605, Cambridge, Abcam), anti-MMP2 (ab37150, Abcam), anti-MMP7 (ab5706, Abcam), anti-MMP9 (ab38898, Abcam), anti-Slug (ab27568, Abcam), anti-Twist (ab50887, Abcam) and anti-GAPDH (ab8245, Abcam). Chemiluminescence detection system (GE Healthcare, Chicago, IL, USA) was employed to measure the protein.
10
Immunofluorescence Analysis of Ovarian Cancer Markers
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Ovarian cancer cells were incubated with blocking buffer (5% normal goat serum, 3% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h. Primary antibodies to YAP1 (ab205270; 1:200; Abcam), E-cadherin (ab76055; 1:150; Abcam), vimentin (ab45939; 1:100; Abcam), Slug (ab27568; 1:150; Abcam), and PCNA (ab29; 1:100; Abcam) were incubated with the cells overnight. After three rinsing for 5 min with PBST, Alexa Fluor 568 (red, goat anti-rabbit), 488 (green, goat anti-rabbit) and 488 (green, goat anti-mouse) antibodies were applied (1200 dilution, Invitrogen, Thermo Fisher, USA) for 1 h at room temperature. Cell nuclei were counterstained with DAPI (Burlingame, CA). Images were taken using a Nikon Ti inverted fluorescence microscope.
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