Cells were harvested at the logarithmic growth phase after overexpression treatment. The obtained cells were lysed in RIPA buffer supplemented with PMSF (RIPA: PMSF ratio of 99:1, Solarbio, Beijing, China), and total protein was quantified with the bicinchoninic acid (BCA) method (Biorad, China). The extracted proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto nitrocellulose membranes, blocked with non-fat milk (BD, USA) for 1 h at room temperature, and then incubated with primary antibodies against DDR1 (Catalog #sc-21790, Abcam), COL4A1 (eBioscience, Catalog# ab86042, Abcam), MMP-2 (Catalog # ab97779, Abcam), SLUG (Catalog #ab97779, Abcam), ZEB1 (Catalog #ab203829, Abcam), and β-actin polyclonal antibody (Catalog # ab179467, Abcam). β-actin was used as a control. The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies rabbit anti-Mouse IgG H&L (1:5000, ab6728; 1:5000; Abcam) or donkey anti-rabbit IgG H&L (1:5000, ab6802, Abcam, USA). Using enhanced chemiluminescence (ECL) chromogenic substrate (Thermo Fisher), the antibody-reactive protein bands were visualized by the SuperSignal™ West Dura system (Pierce Biotechnology, Inc., Rockford, IL, USA), and UVITech imager (UVITech, Inc., Cambridge, UK) was used to photograph the membrane.
Ab97779
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab97779 is a laboratory equipment product. It serves as a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab38898, by Abcam (27 mentions)
Pvdf membrane, by Merck Group (14 mentions)
Ab9485, by Abcam (13 mentions)
Ab205718, by Abcam (9 mentions)
Bca kit, by Thermo Fisher Scientific (9 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
Ab76003, by Abcam (6 mentions)
Ab92552, by Abcam (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Ripa buffer, by Beyotime (5 mentions)
Ab18203, by Abcam (4 mentions)
Ab137867, by Abcam (4 mentions)
Image lab software, by Bio-Rad (4 mentions)
Ab32503, by Abcam (4 mentions)
Ab32124, by Abcam (4 mentions)
Ab8227, by Abcam (4 mentions)
Ab2302, by Abcam (4 mentions)
Ab8245, by Abcam (4 mentions)
Ab16663, by Abcam (4 mentions)
78 protocols using ab97779
1
Western Blot Analysis of EMT Markers
2
Protein Expression Analysis in Ovarian Cancer
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Total protein from OC tissues and cells was extracted using protein extraction lysate (Beyotime, Shanghai, China). Then, protein concentration was evaluated by BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were separated by 10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). Next, PVDF membranes were blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with primary antibody against CDCA7 (Thermo Fisher Scientific, PA5-101,299, 1:1000), EZH2 (Thermo Fisher Scientific, MA5-15,101, 1:1000), CyclinE1 (Abcam, ab33911, 1:1000), CyclinE2 (Abcam, ab40890, 1:5000), MMP2 (Abcam, ab97779, 1:2000), MMP9 (Abcam, ab228402, 1:1000), VEGFA (Abcam, ab214424, 1:1000), VEGFR1 (Abcam, ab32152, 1:5000), VEGFR2 (Abcam, ab134191, 1:5000) and GAPDH (Thermo Fisher Scientific, MA5-15,738-D680, 1:1000). The next day, membranes were washed with TBST and incubated with the corresponding secondary antibody (Thermo Fisher Scientific, A32728, 1:10,000) for 2 h at room temperature. Enhanced chemiluminescence reagents (ECL; Pierce, IL, USA) were applied to visualize the protein bars. Finally, protein bands were detected by a Bio-Rad imaging system (Bio-Rad, CA, USA).
3
Western Blot Analysis of Protein Expression
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Total protein of H1299 and A549 cells was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime). Proteins were subsequently separated by electrophoresis using SDS‐PAGE gel and then transferred onto polyvinylidene difluoride (PVDF) membrane (Beyotime). The membrane was maintained overnight with primary antibodies including cyclin D1 (1:10 000, ab134175), MMP2 (1:1000, ab97779), NFAT5 (1:500, ab137407) or GAPDH (1:10 000, ab181602) that were purchased from Abcam. Afterwards, these membranes were incubated with secondary antibodies (ab97051, 1:5000, Abcam). The protein blots were visualized by using enhanced chemiluminescence (ECL) kits (Solarbio).
4
Tumor Immunohistochemistry Protocol
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Tumor masses were fixed and embedded in paraffin. Then, 8% bovine serum albumin (BSA) solution was used to block tumor specimens. Tumor sections were then incubated with primary antibodies against NFAT5 (1:50, ab3446, Abcam), Ki67 (1:200, ab15580, Abcam) and MMP2 (1:500, ab97779) and secondary antibody (ab97051, 1:1000). Finally, the sections were imaged with a light microscope (Leica).
5
Immunohistochemical Staining of Brain Sections
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Immunohistochemical staining was performed as previously described (Miao et al. 2020 (link)). Briefly, coronal brain sections were blocked with 5% goat serum in phosphate-buffered saline with 0.1% Triton-X 100 for 1 h, followed by primary antibody incubations for 1 h at room temperature and overnight incubation at 4 °C. The following primary antibodies were used: Rat anti MAP2 (1:400; Millipore), Rabbit anti Ly6G (1:300; Abcam), Rabbit anti F4/80 (1:300; Bio Legend), Alexa Flour 488-conjugated (1:300; Invitrogen), Rat anti CD31 (1:300; BD Biosciences). Rabbit anti-MMP-2 (1:200, ab97779, Abcam), goat anti-CD206 (1:250, AF2535, R&D), rat anti-CD16/32 (1:250, ab25235, Abcam), and rabbit anti-Iba1 (1:1000, ab5076, Abcam).
6
Western Blot Analysis of Signaling Proteins
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Protein extracts were prepared in radio immunoprecipitation assay (RIPA) lysis buffer (Sigma), resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Blocking was performed using 5% non-fat milk, followed by incubation with corresponding primary and secondary antibodies. Primary antibodies from Abcam (Cambridge, UK) against RASSF1A (1:500, ab97749), GSK-3β (1:500, ab131356), p-GSK-3β (1:500, ab75745), β-catenin (1:2,000, ab16051), MMP-2 (1:1,000, ab97779), MMP-9 (1:300, ab38898) and p65 (1:1,000, ab16502) were obtained. It was normalized with β-actin (1:1,000, ab8227) and visualized by chemiluminescence.
7
Western Blot Analysis of Cell Signaling Proteins
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The cells were lysed using RIPA buffer (08714-04, Nacalai Tesque Inc., Kyoto, Japan) and the protein concentration of samples was quantified using BCA assay. Equal amounts of proteins (120 μg) were separated by SDS-PAGE and the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk for 30 mins, and then incubated overnight at 4° C with primary antibodies against BATF (ab236876, dilution 1:1000; Abcam, Cambridge, MA, USA), TGFβ1 (ab92486, 1:1000; Abcam, Cambridge, MA, USA), CD147 (ab188190, dilution 1:5000; Abcam, Cambridge, MA, USA), MMP-2 (ab110258, dilution 1:1000; Abcam), MMP-9 (ab97779, dilution 1:1000; Abcam, Cambridge, MA, USA), and GAPDH (5174S, dilution 1:2000; Cell Signaling Technology, Inc. Danvers, CO, USA). Then, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (ab6789 or ab6721, dilution 1:500, Abcam) for 30 min at room temperature. The blots were developed and visualized using ECL Plus Western Blotting Detection kit (GE Healthcare, Buckinghamshire, UK).
8
Protein Expression Analysis in Cancer Cells
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LoVo or SW1116 cells were lysed on ice with the lysis buffer (Beyotime, Shanghai, China) and the lysates were later centrifuged for 15 min at 12,000 × g. The BCA kit (Thermo Fisher Scientific, Waltham, MA, USA) was acquired for evaluating protein concentrations. Protein was subjected to SDS–PAGE (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Primary antibodies and secondary antibodies were applied to incubate membranes in sequence. ECL detection system (Applied Biosystems, Foster City, CA, USA) was utilized to visualize protein bands. Primary antibodies against MMP2 (ab97779, Abcam, Cambridge, MA, USA), MMP7 (ab5706, Abcam), N-cadherin (ab76057, Abcam), E-cadherin (ab40772, Abcam), NANOG (ab80892, Abcam), OCT4 (ab181557, Abcam), Gli4 (AV37797, Sigma-Aldrich), PTCH1 (ab53715, Abcam), Shh (ab53281, Abcam), Gli1 (ab49314, Abcam), Gli2 (ab167389, Abcam), and GAPDH (ab9484, Abcam) were used, individually.
9
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Beyotime) was used for protein extraction. Then protein was treated according to the previous description [12 ]. The primary antibodies were anti-matrix metalloproteinase 2 (MMP2; ab97779; Abcam, Cambridge, MA, USA), anti-matrix metalloproteinase 9 (MMP9; ab76003; Abcam) and anti-FZD5 (ab75234; Abcam). Anti-GAPDH (ab9485; Abcam) was used as a loading control.
10
Protein Expression Analysis of Key Signaling Pathways
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Total protein was extracted from the indicated cells or tissues using RIPA lysis buffer (Beyotime, China), and the protein concentration was measured using a BCA protein assay kit (Beyotime, China). The protein was separated by 10% SDS-PAGE and subjected to western blotting, as described previously [43 (link)]. The primary antibodies used in western blotting were as follows: LIMK1(1:1000, ab81046, Abcam), p-cofilin (1:1000, #3313, CST), cofilin (1:1000, #5175, CST), p-CREB(Ser133) (1:1000, #9198, CST), CREB (1:1000, #9197, CST), MMP2(1:1000, ab97779, Abcam), ITGB1(1:1000, ab134179, Abcam) and COL1A1(1:1000, #72026, CST); GAPDH (1:1000, Boster, Wuhan, China) was used as a loading control. Horseradish peroxidase–conjugated anti-mouse or anti-rabbit IgG antibodies (1:3000, Boster, Wuhan, China) were used as secondary antibodies, and the protein expression levels were visualized using an enhanced chemiluminescence system (Bio-Rad, Hercules, EDAUSA). Band intensities from three biological experiments were quantified by densitometry using ImageJ software.
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