Slides with cryosections were dried up at room temperature and then boiled in the boiling buffer (800 ml milli-Q water, 4 ml 1 M Tris pH8, 1.6 ml 0.5 EDTA) for antigen recovery. Then the brain sections were blocked in 150 μl blocking buffer (0.1 M PBS; 10% NGS and 0.1% Tween 20) and incubated overnight with the following primary antibodies: 1:300 Anti-Nras, Abcam, ab77392; 1/500 Anti-GFP, Proteintech, 50430-2-AP; 1/500 Anti-mCherry, Abclonal, AE002. Signals were visualized using the 1:400 diluted secondary antibody conjugated with AlexaFluor-488, -561 or −647 (Proteintech and Abclonal). After nucleic DNA staining by 4′,6-diamidino-2-phenylindole (DAPI, Sigma), slices were mounted with fluorescent anti-fade mounting medium (Dako). Images were acquired using the Ni-E A1 HD25 confocal microscope (Nikon).
Anti gfp
Manufactured by Proteintech
Sourced in United States, China, Germany, United Kingdom
The Anti-GFP product is a specific antibody designed to detect and bind to the Green Fluorescent Protein (GFP). This antibody can be used in various techniques, such as immunoprecipitation, Western blotting, and immunocytochemistry, to identify and quantify GFP-labeled proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (18 mentions)
Anti flag, by Proteintech (7 mentions)
Anti ha, by Roche (7 mentions)
Anti gapdh, by Proteintech (6 mentions)
Aj1236b, by Thermo Fisher Scientific (6 mentions)
Anti β actin, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (4 mentions)
Anti ha, by Proteintech (4 mentions)
Anti rat hrp, by Thermo Fisher Scientific (4 mentions)
Anti histone h3, by Abcam (3 mentions)
Anti myc, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Anti flag antibody, by Proteintech (3 mentions)
Anti β actin, by Proteintech (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Anti rgs his, by Qiagen (3 mentions)
Anti myc, by Proteintech (3 mentions)
Anti gst, by Proteintech (2 mentions)
Anti e2f1, by Abcam (2 mentions)
75 protocols using anti gfp
1
Immunofluorescence Staining of Cryosectioned Brain Tissue
2
Western Blot Analysis of Protein Markers
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Protein samples were separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) followed by gel transfer to nitrocellulose membrane (Cytiva). The membranes were sequentially incubated with primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies followed by detection with enhanced chemiluminescence system (Millipore) using Amersham Imager 680 (Cytiva). The primary antibodies used in this study were anti-FLAG (Sigma-Aldrich, F3165, 1:8000 diluted), anti-GAPDH (ImB, MM001, 1:3000 diluted), anti-MeCP2 (Diagenode, pAb-052-050), anti-Rbfox1(Millipore, MABE159), anti-Rbfox2 (Bethyl, A300–864A), anti-Rbfox3 (Millipore, MAB377), anti-Matrin3 (Bethyl, A300–591A), anti-hnRNP M (Santa Cruz, sc-20001), anti-hnRNP A1 (Santa Cruz, sc-32301), anti-hnRNP C (Abclonal, A0057), anti-hnRNP H1 (Abclonal, A5924), anti-hnRNP U (Abclonal, A9907), anti-hnRNP F (Abclonal, A5505), anti-NF110 (Abclonal, A2496), anti-hnRNP K (Abclonal, A1701), anti-histone H3 (Abcam, ab1791), anti-DDX5 (Abcam, ab21696), and anti-GFP (Proteintech, 66002-1-Ig). Dilutions of all primary antibodies were 1:1000 if not specifically mentioned. HRP-conjugated secondary antibodies (1:2500 diluted) were anti-mouse IgG (Promega, W4021), anti-rabbit IgG (Promega, W4011), and conformation-specific anti-rabbit IgG (Cell Signaling, 3678).
3
Cell Line Characterization Using Antibodies
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DMSO, G418, and doxycycline hyclate (DOX) were purchased from Nacalai Tesque (Kyoto, Japan), Adipogen Life Sciences (Liestal, Switzerland), and Cayman Chemical Company (Ann Arbor, MI, USA), respectively. The following antibodies were used: anti‐FLAG (Sigma‐Aldrich, St. Louis, MO, USA), anti‐HA (Roche Applied Science, Penzberg, Germany), anti‐Myc (Santa Cruz Biotechnology, Inc, Dallas, TX, USA), anti‐Arp5 (Proteintech, Rosemont, IL, USA), anti‐GAPDH (Thermo Fisher Scientific, Waltham, MA, USA), anti‐ACTA2 (Sigma‐Aldrich), anti‐Collagen I (Novus Biologicals, Centennial, CO, USA), and anti‐GFP (Proteintech) antibodies.
4
Antibody-based Characterization of Tubulin and Centromeres
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Anti-α-tubulin (mouse, DM1A, Sigma 05-829) and ACA (anti-centromere antibody, Immunovision HCT-0100) were used for immunofluorescence. Anti-CENP-N antibody were kindly gifted by Dr. Iain Cheeseman. Antibodies used for Western blots were anti-α-tubulin (mouse, DM1A, Sigma 05-829, 1:5,000), anti-β-Actin (Servicebio, GB12001), anti-FLAG-tag (Sigma F1804, 1:2,000), anti-GFP (Proteintech, 50430-2-AP, 1:1,000). The appropriate secondary antibodies were purchased from Jackson ImmunoResearch Laboratories and used as instructed by the vendor’s instruction.
5
Immunoprecipitation and Western Blotting Protocol
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Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described [37 ]. Nuclear proteins were prepared with the NE-PER Kit (Pierce) following manufacturer's recommendation. Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads (M2, Sigma) were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3X FLAG peptide (Sigma). Western blotting was performed with anti-FLAG (Sigma, F3165), anti-GFP (Proteintech, 50430-1), anti-MKL1 (Proteintech, 21166-1), anti-E2F1 (Cell Signaling Tech, 3742), anti-FOXM1 (Abcam, ab207298), anti-phosphorylated serine/threonine (Cell Signaling Tech, 9631), anti-MK2 (Cell Signaling Tech, 3042), anti-phosphorylated MK2 (Cell Signaling Tech, 3316), and anti-β-actin (Sigma, A2228).
6
Immunoprecipitation and Immunoblotting Protocol
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Cells were harvested and lysed respectively in IP Lysis Buffer (Thermo, USA) supplemented with protease and phosphatase inhibitors (Roche, Switzerland). The protein samples were incubated with indicated antibody in 1 mL IP Lysis Buffer overnight at 4 °C, and then were precipitated with 20 μL Protein A/G Plus-agarose (Roche, Switzerland). After a brief centrifugation, the pellet was washed 3 times with IP Lysis Buffer. The lysates and immunoprecipitates were analyzed by immunoblotting.
Immunoblotting was performed using indicated primary antibodies: anti-MAGE-G1 (B-Bridge, USA), anti-GFP (Proteintech, USA), anti-GAP43 (Sigma-Aldrich, USA), anti-Neuron-specific III β-tubulin (Bioworld, China), anti-GAPDH (Bioworld, China), anti-active Caspase3 (Sigma-Aldrich, USA), anti-FSCN1 (Sigma-Aldrich, USA) and anti-VIME (Sigma-Aldrich, USA), anti-GST (Proteintech, USA), anti-Flag (MBL, USA). Detailed information of immunoblotting analysis was previously described42 .
Immunoblotting was performed using indicated primary antibodies: anti-MAGE-G1 (B-Bridge, USA), anti-GFP (Proteintech, USA), anti-GAP43 (Sigma-Aldrich, USA), anti-Neuron-specific III β-tubulin (Bioworld, China), anti-GAPDH (Bioworld, China), anti-active Caspase3 (Sigma-Aldrich, USA), anti-FSCN1 (Sigma-Aldrich, USA) and anti-VIME (Sigma-Aldrich, USA), anti-GST (Proteintech, USA), anti-Flag (MBL, USA). Detailed information of immunoblotting analysis was previously described42 .
7
Protein Detection by Western Blot
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Cell lysates were prepared 48 h after transfection and were separated by SDS–12% PAGE, followed by transfer to nitrocellulose. Protein detection was carried out using standard protocols with anti-HA antibody (66006; Proteintech), anti-GFP (66002; Proteintech), anti-FLAG (F1804; Sigma), and anti-actin (sc-47778; Santa Cruz Biotechnology). Luminescent signals were detected with the ChemiDoc MP imaging system (Bio-Rad).
8
Co-Immunoprecipitation of STAG3, REC8, and SMC1A
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Co-IP was performed as described previously (Wolf et al., 2018 (link)). Briefly, HEK293 cells were transfected with expression vectors containing wild-type FLAG3-STAG3 and REC8-EGFP plasmids (or SMC1A-HA plasmids) or mutated FLAG3-STAG3 and REC8-EGFP plasmids (or SMC1A-HA plasmids), according to the manufacturer’s instructions for Lipofectamine 3000 use. Untransfected HEK293 cells were used as a negative control. After incubation for 36 h, the culture medium was supplemented with 0.2 µg/ml nocodazole (Sigma, Louis, MO, USA) for prometaphase arrest of the cells. Total protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific) after 12 h, and then, western blotting was performed to detect the expression of STAG3, REC8, and SMC1A using anti-FLAG, anti-GFP, and anti-HA (Proteintech) antibodies, respectively, at a 1:1,000 dilution. Next, immunoprecipitation was performed using Dynabeads Protein G (Invitrogen) following the manufacturer’s instructions.
9
Characterization of DAPK and miRNA pathway
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HeLa and 293Tcells were cultured in MEDM plus 10% FBS. Both cells were determined to be free from mycoplasma contamination. The antibodies used were from different vendors: Anti-DAPK1 (Abcam, ab109382; ABclonal, A5741), Anti-DAPK2 (Abgent, AP7033A), Anti-DAPK3 (Abgent, AJ1236b; Thermal scientific, PA5–27700), Anti-ACTIN (PTGCN, 60008-I-Ig), Anti-E2F1 (Abcam, ab179445), Anti-GW182 (Bethyl, A302–329A), Anti-TNRC6B (Abnova), Anti-TNRC6C (Bethyl, A303–969A), Anti-Ago1 (Abcam, ab105104), Anti-Ago2 (Abnova, H00027161-M01), Anti-Ago3 (Proteintech, 19692-I-AP), Anti-Ago4 (CST, 6913S), Anti-CD99 (Proteintech, 60354-I-Ig; ABclonal, A2028), Anti-NDRG1 (Abcam, ab124698), Anti-SAFB (Abnova, H00006294-M04), Anti-FLAG (ABclonal, AE005), Anti-GFP (Proteintech, 66002-I-Ig), anti-Mcherry (ABclonal, AE002). Western blotting results were quantified by the ImageJ software.
10
Protein expression and western blotting analysis
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Stable overexpression cells were grown up to 90% confluency in 6 well plates and lysed in RIPA buffer with 1 mM DTT, 1 mM benzamidine, 1 mM PMSF and 1× protease inhibitor cocktail. Cell resuspensions were incubated for 15 min on ice, followed by short sonication. The supernatants of lysates were collected after centrifugation at 10,000 rpm for 10 min at 4 °C. The samples were heated with 1× SDS loading buffer at 98 °C for 5 to 10 min. Following 10% or 15% SDS-PAGE and wet blot transfer, the blots were probed with one of the following primary antibodies in TBST: monoclonal anti-FLAG® M2 antibody (Merck KGaA, Cat. No. F1804, Darmstadt, Germany), mTOR (phosphor-S2448) polyclonal antibody (Bioworld, Cat. No. BS4706, Nanjing, China), mTOR (S2442) polyclonal antibody (Bioworld, Cat. No. BS3611, Nanjing, China), anti-HIF-1α (Sangon, Cat. No. D162108, Shanghai, China); anti-LC3 (abcam, Cat. No. ab51520, Cambridge, UK); anti-p62 (abcam, Cat. No. ab56416, Cambridge, UK); anti-GFP (Proteintech, Cat. No. 66002-1-Ig, Wuhan, China), anti-GAPDH (Proteintech, Cat. No.60004-1-Ig, Wuhan, China).
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