Cultured cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China). BCA kit (Pierce, Rockford, IL) was used to measure protein concentrations. The Flag and HA sequences were inserted into plasmids by the primers containing specific restriction sites. The plasmids were transformed into competent cells, screened, and amplified with LB containing ampicillin. High-concentration recombinant Flag-TMED3 and HA-FAM60A were obtained. These two plasmids were verified by DNA sequencing. Endotoxin-free extraction was carried out for subsequent cell transfection [18 (link)]. The equivalent amounts of proteins were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA). PVDF membrane was blocked with a blocking buffer and was incubated with an anti-TMED3 (ab223175, Abcam), anti-FAM60A (ab167180, Abcam), anti-GAPDH (AP0063, Bioworld, Nanjing, China), anti-DYKDDDDK Tag (14,793, Cell Signaling, Danvers, MA), anti-HA (ab9110, Abcam), anti-GAPDH antibody (AP0063, Bioworld), and the corresponding secondary antibodies, goat anti-rabbit IgG (A0208, Beyotime) or goat anti-mouse IgG (A0216, Beyotime). Immobilon Western Chemiluminescent HRP Substrate kit (Millipore) was used for color development and the bands were detected using an Amersham Imager 600 (GE Healthcare Little Chalfont, Buckinghamshire, UK).
Ap0063
Manufactured by Bioworld Technology
Sourced in United States, China
AP0063 is a laboratory instrument designed for the extraction and purification of nucleic acids, such as DNA and RNA, from various biological samples. The core function of this equipment is to automate the extraction process, ensuring consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Gapdh, by Bioworld Technology (5 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Anti gapdh, by Bioworld Technology (4 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Bs12478, by Bioworld Technology (2 mentions)
Quantity one, by Bio-Rad (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ab9722, by Abcam (2 mentions)
Hpa001562, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Ab32503, by Abcam (2 mentions)
Af887 sp, by R&D Systems (2 mentions)
Whole protein extraction kit, by Keygen Biotech (2 mentions)
Ab151247, by Abcam (2 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
56 protocols using ap0063
1
Protein Expression and Immunoblotting
2
Western Blot Analysis of c-Kit Protein
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Fresh-frozen antrum specimens were homogenized in extraction buffer, and were centrifuged at 12,000 g for 10 min at 4°C, and protein concentration in the supernatant was quantified by the bicinchoninic acid (BCA) method. Later, equivalents of 40μg of extracted proteins were separated using 12% SDS-PAGE, and the separated proteins were then transferred electrophoretically onto PVDF membranes. After blocking nonspecific binding sites with 5% nonfat dry milk in Tris·HCl-buffered saline (TBS) for 1 h, the membranes were then incubated with primary antibodies to c-Kit (1:200,sc-168,Santa, CA.), respectively, overnight at 4°C. Anti-rat GAPDH (1:500,AP0063, Bioworld Technology, Inc.) served as the internal control. After that, the membranes were washed in TBST (TBS with 0.1% Tween-20) for three times and incubated with HRP-linked secondary antibody (1:5,000,Goat anti-Rabbit IgG-HRP,Bioworld Technology, Inc.) for 1 h at room temperature. Detection of protein was achieved by ECL reagents, and the blot was subjected to autoradiography. A semiquantitative measurement of the band intensity was performed by Quantity One (ChemiDoc XRS+ System, Bio-RAD,Inc.)
3
Quantitative Western Blot Analysis of POSTN
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Rabbit monoclonal antibody (mAb) to human POSTN (1:1000, ab172615, Abcam, UK), mouse mAb to human Phospho-Akt (Ser473) (587F11) (1:1000, 4051, Cell Signaling Technology, Beverly, MA), rabbit polyclonal antibody (pAb) to human Akt (1:1000, 9372, Cell Signaling Technology), rabbit mAb to human β-catenin (1:1000, 8480, Cell Signaling Technology) and rabbit pAb to human GAPDH (1:1000, AP0063, Bioworld Technology) were applied for Western blot analysis. Signal intensity of each band was quantified using Genetools software (version 4.02, Cambridge, UK). Relative expression was calculated as that signal intensity of POSTN divided by signal intensity of GAPDH in each lane as previously described [38 (link)].
4
Protein Expression Analysis of Notch Pathway
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The cells at passage 3 were rinsed twice with precooled PBS, then 1 mL of cell lysis buffer (50.0 mmol/L Tris pH = 7.6, 150.0 mmol/L NaCl, 0.1% SDS, 1.0% NP-40, protease inhibitor cocktail) was added, and the cells were scraped off. The cells were lysed at 4°C for 30 min under rotation and centrifuged at 15000 rpm for 30 min, and the supernatant was collected. Protein concentrations were determined by the BCA Protein Assay Reagent (Thermo Fisher Scientific, Rockford, IL, USA), after which 25 μg of total proteins was loaded to 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (PVDF, Millipore) using the conventional method. The membrane was immunoblotted with primary antibodies against CD146 (1 : 500, ab75769, Abcam), Jagged1 (1 : 500, ab109536, Abcam), DLL4 (1 : 500, ab7280, Abcam), or GAPDH (1 : 10000, AP0063, Bioworld technology), followed by incubation with a goat anti-mouse (1 : 10000, BS12478, Bioworld technology) or goat anti-rabbit (1 : 10000, A0545, Sigma) secondary antibody. The bands were detected using an enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA), and densitometric analysis of each band was performed with Quantity-one (Bio-Rad) software.
5
Quantitative Protein Expression Analysis
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Primary astrocytes or brain tissues were lysed to collect proteins. Equal amounts of proteins were electrophoresed on SDS-PAGE, transferred to a PVDF membrane, and then blocked at room temperature for 2 h. Membranes were incubated with primary antibodies against anti-GS (1:2000 dilution, Abcam, ab64613), anti-VDAC1 (1:1000 dilution, Abways Technology, CY5416), anti-FLAG (1:8000 dilution, Bioworld Technology, AP0007 M) and anti-GAPDH (1:8000 dilution, Bioworld Technology, AP0063). After blotted with the primary antibodies at 4 °C overnight, all the membranes were incubated for 2 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. The values of band intensities were detected by enhanced chemiluminescence (ECL) and quantized by Image-Pro Plus 6.0 software.
6
Western Blot Analysis of KLF12, Nur77, and GAPDH
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Proteins were extracted as described previously [17 (link)]. The protein concentrations were measured by Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts (25 μg) of protein were separated on a 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblotting was performed by incubating the membranes with primary antibodies against KLF12 (1:2000; sc-84347, rabbit Polyclonal Antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nur77 (1:1000; 3960, rabbit Monoclonal Antibody, Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:10000; AP0063, GAPDH polyclonal antibody, Bioworld Technology, MN, USA), followed by incubation with a goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000; BS13278, Bioworld Technology, St. Louis Park, MN, USA) and Flag-HRP (1:5000; A8592, Sigma, St. Louis, MO, USA). Detection was performed using an enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA), and densitometric analysis of each band was performed with Quantity-one (Bio-Rad, Hercules, CA, USA) software.
7
Western Blot Analysis of Notch Signaling
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Cells were collected and lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher scientific). The protein was separated on 10% polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes. After being blocked, the membranes were incubated at 4°C with the following primary antibodies and followed by a secondary antibody at room temperature. Dilution of primary antibodies and secondary antibodies was as follows: Notch1 (1:500; ab27526; Abcam; Eugene, OR, USA), Notch2 (1:1,000; 5732S; Cell signal Technology; Danvers, MA, USA), Notch3 (1:1,000; 5276; Cell signal Technology), Notch4 (1:5,000; 2423; Cell signal Technology), DLL3 (1:1,000; ab103102; Abcam), Hes1 (1:1,000; D6P2U; Cell signal Technology), GAPDH (1:30,000; AP0063; Bioworld Technology; St Louis, MN, USA), Goat anti-Rabbit IgG-HRP (1:10,000; FDR007; Fdbio science; Hangzhou, Zhejiang, China), and Goat anti-Mouse IgG-HRP (1:5,000; FDM007; Fdbio science). The bands of proteins were confirmed by luminescent visualization using an ECL Western Blotting Detection System (Bio-Rad, Hercules, CA, USA) and quantified using the Quantity One software package (Bio-Rad).
8
Immunoblot Analysis of Cellular Proteins
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Immunoblot was carried out as described in a previous report (Gao, 2016 b). In brief, following treatment with RIPA lysis buffer (P0013, Beyotime, China), total protein was quantitated with a BCA protein assay kit (P0012, Beyotime).
After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein bands underwent transfer onto polyvinylidene fluoride (PVDF) membranes. This was followed by overnight incubation (4℃) with primary antibodies raised against integrin α1 (1:1,000; sc-271,034, Santa Cruz, USA), caveolin-1 (1:1,000; sc-894, Santa Cruz), caveolin-2 (1:1,000; ab2911; Abcam, UK), caveolin-3 (1:1,000; ab2912; Abcam), and GAPDH (1:5,000; AP0063; Bioworld Technology, USA). Following washing with PBS, goat anti-mouse (GAM007; MultiSciences Technology, USA) or anti-rabbit (GAR0072; MultiSciences Technology) IgG-HRP was added for 1 h at ambient. Finally, Immobilon™ Western Chemiluminescent HRP substrate reagent (EMD Millipore, USA) was employed for development. Immunoreactive bands were captured and assessed on a Bio-Rad Gel Doc Imaging System (Bio-Rad Laboratories, USA).
After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein bands underwent transfer onto polyvinylidene fluoride (PVDF) membranes. This was followed by overnight incubation (4℃) with primary antibodies raised against integrin α1 (1:1,000; sc-271,034, Santa Cruz, USA), caveolin-1 (1:1,000; sc-894, Santa Cruz), caveolin-2 (1:1,000; ab2911; Abcam, UK), caveolin-3 (1:1,000; ab2912; Abcam), and GAPDH (1:5,000; AP0063; Bioworld Technology, USA). Following washing with PBS, goat anti-mouse (GAM007; MultiSciences Technology, USA) or anti-rabbit (GAR0072; MultiSciences Technology) IgG-HRP was added for 1 h at ambient. Finally, Immobilon™ Western Chemiluminescent HRP substrate reagent (EMD Millipore, USA) was employed for development. Immunoreactive bands were captured and assessed on a Bio-Rad Gel Doc Imaging System (Bio-Rad Laboratories, USA).
9
Antibody-based Protein Detection in Cell Studies
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The specific antibodies against the followings were used in this study: hSETD8 (ab3798 from Abcam, Cambridge, MA, USA, for ChIP; 06‐1304 from Millipore, Billerica, MA, USA, for immunoblotting), H4K20me1 (ab9051 from Abcam), p53 (sc126 from Santa Cruz Biotechnology, Dallas, TX, USA), p21 (05‐345 from Millipore), GAPDH (AP0063 from Bioworld Technology, St Louis Park, MN, USA), and PPARγ (ab41928 from Abcam, for ChIP; 81B8 from Cell Signaling, Danver, MA, USA, for immunoblotting). Western blot analysis was performed after electrophoretic separation of polypeptides by SDS‐PAGE and transfer to Immobilon‐P/PVDF membrane (Millipore). Blots were probed with the indicated primary and appropriate secondary antibodies. Immunobands were subsequently detected by the enhanced chemiluminescence reaction (ECL) (PerkinElmer; Waltham, MA, USA).
10
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from skin scar tissues utilizing radioimmunoprecipitation assay lysis buffer (Shanghai Shenergy Biocolor BioScience & Technology Company, Shanghai, China). The bicinchoninic acid assay (Thermo Fisher Scientific, Inc.) was used to determine the concentration of protein. The proteins at the dose of 20 µg were isolated by 10% SDS-PAGE and electroblotted in the membranes of polyvinylidene difluoride (EMD Millipore, Bedford, MA, USA). The aspecific protein binding sites on the membranes were blocked with 5% BSA (Biosharp, Hefei, China). Then the membranes were gently agitated and incubated with antibodies against IL-1β (ab9722; 1:500; Abcam), IL-6 (ab208113; 1:500; Abcam), TGF-β1 (ab92486; 1:500; Abcam), TNF-α (ab6671; 1:500; Abcam), GAPDH (AP0063; 1:10,000; Bioworld Technology, Inc., St. Louis Park, MN, USA;), and α-SMA (ab5694; 1:500; Abcam) at 4°C overnight. The following day, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG secondary antibody (7074S; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA). The bands were visualized using an enhanced chemiluminescence detection system (EMD Millipore, Billerica, MA, USA) and quantitative analysis was conducted using Image J software version 1.4.3.67 (National Institutes of Health, Bethesda, MD, USA).
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