For quantification of specific GFP positive cell populations, the following triple labeling was performed: mature neurons—mouse anti-GFP (1:200; Santa Cruz Biotechnology), chicken anti-β-III tubulin (1:100; Millipore), and rat anti-BrdU (1:400; Accurate Chemical & Scientific Corp.); neuroblasts—mouse anti-GFP, goat anti-doublecortin (DCX; 1:400; Santa Cruz Biotechnology), and rat anti-BrdU; progenitor cells—mouse anti-GFP, rat anti-BrdU, rabbit anti-nestin (1:200; Millipore); stem cells- goat anti-GFP, mouse anti-nestin, rabbit anti-GFAP (1:500; Dako); mature astrocytes—goat anti-GFP, mouse anti-GFAP, rabbit anti-S100ß (1:3000, Abcam). The following secondary antibodies were used from Jackson ImmunoResearch Laboratories (West Grove, PA): biotinylated species-specific anti-IgG (all used at 1:250), Cy3-conjugated Donkey anti-Rat (1:500), Alexa Fluor 647 (AF647)-conjugated Donkey anti-Mouse (1:250), Cy2-conjugated Streptavidin (1:250), DAPI Nucleic Acid Stain (1:10,000, Life Technologies, Grand Island, NY).
Mouse anti gfp
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-GFP is a monoclonal antibody that specifically recognizes and binds to the Green Fluorescent Protein (GFP). It is a powerful tool for the detection and localization of GFP-tagged proteins in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti flag, by Merck Group (7 mentions)
Rabbit anti gfap, by Agilent Technologies (4 mentions)
Mouse anti α tubulin, by Merck Group (4 mentions)
Mouse anti β actin, by Merck Group (4 mentions)
Mouse anti actin, by Merck Group (3 mentions)
Mouse anti flag m2, by Merck Group (3 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (3 mentions)
Goat anti doublecortin dcx, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti β actin, by Santa Cruz Biotechnology (2 mentions)
Mouse anti ha, by Fortrea (2 mentions)
Mouse anti myc, by Santa Cruz Biotechnology (2 mentions)
Mouse anti ha, by Santa Cruz Biotechnology (2 mentions)
Mouse anti calbindin, by Merck Group (2 mentions)
Mouse anti trrap, by Euromedex (2 mentions)
Rabbit anti stmn3, by Proteintech (2 mentions)
Mouse anti v5, by Thermo Fisher Scientific (2 mentions)
Gel electrophoresis reagents, by Bio-Rad (2 mentions)
Vectashield with 4 6 diamidino 2 phenylindole, by Vector Laboratories (2 mentions)
Alexa 647, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (2 mentions)
61 protocols using mouse anti gfp
1
Quantifying GFP-Positive Cell Populations
2
Characterization of DGCR2 in Neuronal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chemicals were purchased from Sigma-Aldrich unless otherwise indicated. Information of primary antibodies is as follows: mouse anti-FLAG (Sigma, F1804, 1:5000 for WB), mouse anti-GFP (Santa Cruz, sc-9996, 1:1000 for WB), mouse anti-GFP (Invitrogen, A-11,120, 1:1000 for staining), mouse anti-PSD95 (Millipore, MAB1598, 1:1000 for WB and 1:500 for staining), mouse anti-synaptophysin (Dako, M7315, 1:5000 for WB), rabbit anti-β-actin (Santa Cruz, sc-1616-R, 1:1000 for WB), mouse anti-Tau-1 antibody (Millipore, MAB3420, 1:500 for staining), mouse anti-MAP2 antibody (Millipore, MAB3418, 1:500 for staining), DGCR2 antibody was generated against hDGCR2-ICD in rabbit (1:1000 for WB and 1:200 for staining).
To generate FLAG-hDGCR2, the human DGCR2 cDNA encoding 22–550 amino acids of DGCR2 without signal peptide was amplified by PCR and subcloned into pFLAG-CMV1 (Sigma, E7273) downstream of an artificial signal peptide sequence and a FLAG epitope. Different cDNAs encoding ΔECD and ΔICD of DGCR2 were amplified with primers 5’- GAAGATCTGATGCGCCTGGTCGTC-3’ and 5’- ACGCGTCGACCTACACCACAGTATTG-3’, 5’-GAAGATCTGCGGCCAGAGCTG-3’ and 5’- ACGCGTCGACCTACCGGTGGACCATGAAG-3’, and subcloned into pFLAG-CMV1 separately. NRXNs and NLGNs constructs were obtained as described previously [48 (link)]. The authenticity of all constructs was verified by DNA sequencing and western blotting analysis.
To generate FLAG-hDGCR2, the human DGCR2 cDNA encoding 22–550 amino acids of DGCR2 without signal peptide was amplified by PCR and subcloned into pFLAG-CMV1 (Sigma, E7273) downstream of an artificial signal peptide sequence and a FLAG epitope. Different cDNAs encoding ΔECD and ΔICD of DGCR2 were amplified with primers 5’- GAAGATCTGATGCGCCTGGTCGTC-3’ and 5’- ACGCGTCGACCTACACCACAGTATTG-3’, 5’-GAAGATCTGCGGCCAGAGCTG-3’ and 5’- ACGCGTCGACCTACCGGTGGACCATGAAG-3’, and subcloned into pFLAG-CMV1 separately. NRXNs and NLGNs constructs were obtained as described previously [48 (link)]. The authenticity of all constructs was verified by DNA sequencing and western blotting analysis.
3
Regulating Nuclear Protein Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa S3, NIH3T3, COS-7 and HEK293T cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and antibiotics at 37°C in a humidified atmosphere with 5% CO2. tsBN2 cells (a kind gift from Mary Dasso, NIH, USA) were regularly grown in DMEM and 10% FBS at 32.5°C (permissive temperature) with 5% CO2. For experiments with depleted RCC1, tsBN2 cells were shifted to 39.5°C (non-permissive temperature) for indicated time points.
Rabbit polyclonal antibodies against Nup358 and GFP have been described earlier [37 (link),38 ]. Rat anti-HA (Roche, 1:100) or mouse anti-HA (Covance, 1:3000) was used for immunostaining. Secondary antibodies used for immunofluorescence were goat anti-rat 350, goat or donkey anti-rabbit 488, donkey anti-mouse 594 (Invitrogen, 1:1000). Hoechst-33342 dye (Sigma-Aldrich) was used to stain the DNA. Western blotting was performed with mouse anti-GFP (Santa Cruz Biotechnology, sc-9996, 1:3,000), mouse anti-Ran (BD biosciences, 610340, 1:10,000), goat anti-RCC1 (Santa Cruz Biotechnology, sc-1161, 1: 3000), mouse anti-CRM1 (BD biosciences, 611832, 1:1500) and mouse anti-α-tubulin (Sigma-Aldrich, T5168, 1:5,000) antibodies.
Leptomycin B (LMB) was purchased from Sigma-Aldrich. After transfection, cells were treated with 5 ng/ml of LMB for indicated time points.
Rabbit polyclonal antibodies against Nup358 and GFP have been described earlier [37 (link),38 ]. Rat anti-HA (Roche, 1:100) or mouse anti-HA (Covance, 1:3000) was used for immunostaining. Secondary antibodies used for immunofluorescence were goat anti-rat 350, goat or donkey anti-rabbit 488, donkey anti-mouse 594 (Invitrogen, 1:1000). Hoechst-33342 dye (Sigma-Aldrich) was used to stain the DNA. Western blotting was performed with mouse anti-GFP (Santa Cruz Biotechnology, sc-9996, 1:3,000), mouse anti-Ran (BD biosciences, 610340, 1:10,000), goat anti-RCC1 (Santa Cruz Biotechnology, sc-1161, 1: 3000), mouse anti-CRM1 (BD biosciences, 611832, 1:1500) and mouse anti-α-tubulin (Sigma-Aldrich, T5168, 1:5,000) antibodies.
Leptomycin B (LMB) was purchased from Sigma-Aldrich. After transfection, cells were treated with 5 ng/ml of LMB for indicated time points.
4
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Triton X‐100 lysis buffer [20 mm Tris (pH 7.4), 2 mm EDTA, 150 mm sodium chloride, 1 mm sodium deoxycholate, 1% Triton X‐100, 10% glycerol, 2 pills protease inhibitor cocktail (Roche)] was used to collect protein from cells. Protein samples were heat‐denatured and equally loaded, separated on 8–12% SDS/PAGE gel, transferred onto a polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK), and blocked with 5% nonfat dry milk. Primary antibodies for western blot analyses included mouse anti‐α‐tubulin (1 : 20 000 dilution; Sigma), rabbit anti‐Snail (1 : 1000 dilution; Cell Signaling, Danvers, MA, USA), rabbit anti‐Slug (1 : 1000 dilution; Cell Signaling), rabbit anti‐CHIP (1 : 1000 dilution; Abgent, San Diego, CA, USA), mouse anti‐Flag (1 : 3000 dilution; Abcam), mouse anti‐GFP (1 : 1000 dilution; Santa Cruz), mouse anti‐HA (1 : 1000 dilution; Abcam, Cambridge, MA, USA), mouse anti‐Vimentin (1 : 1000 dilution; Santa Cruz, Santa Cruz, CA, USA), mouse anti‐E‐cadherin (1 : 5000 dilution; BD Biosciences, San Jose, CA, USA), and mouse anti‐Ub (1 : 5000 dilution; Santa Cruz). Membranes were incubated with horseradish peroxidase‐conjugated anti‐mouse or anti‐rabbit secondary antibody (1 : 5000 dilution; Cell Signaling) for 1 h, and chemiluminescence signals were detected by ECL substrate (GE Healthcare, Chicago, IL, USA).
5
Western Blot Antibody Validation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used for western blot were: mouse anti-Flag (1:5,000, F3165, Sigma), mouse anti-Myc (1:1,000 sc-40, Santa Cruz), mouse anti-HA (1:1,000, sc-7392, Santa Cruz), mouse anti-GFP (1:500, sc-9996, Santa Cruz), mouse anti-α-tubulin (1:1,000, T5168, Sigma), rabbit anti-phospho-histone H3 (Ser 10) (1:5,000, 05-817 R, Millipore), rabbit anti-BubR1 (1:5000, A300-386A, BETHYL), mouse anti-Bub1 (1:1000, B0561, Sigma), rabbit anti-Bub3 (1:1000, ab131157, Abcam), rabbit anti-CENP-E (1:5,000, a gift from Dr. Don W. Cleveland), sheep anti-HOIP and anti-HOIL-1L (1:5,000, a gift from Dr. Philip Cohen), rabbit anti-SHARPIN (1:1,000, #4444, CST), anti-linear Ub62 (link) (1:2500, a gift from Dr. Vishva M. Dixit, Genentech Inc.), rabbit anti-Ub K48 (1:1000, 05-1307, Millipore), rabbit anti-Ub K63 (1:1000, 05-1308, Millipore), rabbit anti-Ska3 (1:3000, a gift from Dr. Hongtao Yu), rabbit anti-KNL1 (1:5000, a gift from Dr. Hongtao Yu). Western blot antibody against SHARPIN of primary MEF cells was: rabbit anti-SHARPIN (1:500, Millipore). Images have been cropped for presentation (uncropped scans of the blots were shown in Supplementary Fig. 7 ).
6
Western Blot Analysis of Transfected Cell Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected cells homogenized in radioimmunoprecipitation assay buffer were centrifuged; then, total protein (20 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Sigma-Aldrich, St. Louis, MO, USA). Non-specific protein binding was blocked by 5% skim milk in 0.05% Tween-20 in Tris-buffer at 37 °C for 1 h; then, the membranes were incubated overnight at 4 °C with mouse anti-GFP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse anti-HCMV IE72 and mouse anti-HCMV IE86 (both from Santa Cruz., Dallas, TX, USA), rabbit anti-RelA/p65, rabbit anti-phospho-RelA/p65 (Ser536), and rabbit anti-p105/p50 (all from Cell Signaling Technology, Danvers, MA, USA), or mouse anti-ß-actin (Sigma-Aldrich) monoclonal antibodies (Table S1 ). Then, the membranes were incubated with horseradish peroxidase-conjugated IgG for 1 h, and proteins were visualized using a chemiluminescent substrate. Band density was quantified using Image J software (http://rsbweb.nih.gov/ij/ , accessed on 8 December 2021) and normalized to the level of β-actin.
7
Protein Extraction and Immunoblotting from Brain and Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein lysates were prepared from brain tissue or aNSCs using the RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate, 1 mM EDTA, 1 mM PMSF), and complete mini protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). N2A cells or neuron lysates were prepared as follows: the culture was treated with 0.25% trypsin for 5 min under 37°C, resuspend with DMEM medium supplemented with 1× FCS and centrifuged under 1100 rpm for 5 min. The resulting pellets were washed once with PBS and lysed with the RIPA buffer. Immunoblotting was performed as described previously (Tapias et al., 2014 (link)), using the following antibodies: mouse anti-Calbindin (1:1000, Sigma), rabbit anti-GFAP (1:1000, Dako-Agilent), mouse anti-CNPase (1:1000, Sigma), rat anti-galectin3 (1:1000, eBioscience, Affymetrix, Santa Clara, USA), mouse anti-α-tubulin (1:5000, Sigma), mouse anti-TRRAP (1;1000, Euromedex, Souffelweyersheim, France), rabbit anti-Sp1 (1:1000, Millipore), mouse anti-RB3/STMN4 (1:1000, Santa Cruz), rabbit anti-STMN3 (1:1000, Proteintech), mouse anti-GAPDH (1:1000, Sigma), mouse anti-β-actin (1:5000, Sigma), mouse anti-GFP (1:400, Santa Cruz), and rabbit anti-Oct4 (1:1000, Cell Signaling).
8
Co-immunoprecipitation Assay for Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in PBS that contained 1% Triton X-100 and a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Cell lysates were then cleared by centrifugation at 12,000× g at 4 °C for 15 min. Alternatively, cells were directly lysed in an electrophoresis sample buffer. For coimmunoprecipitation, GFP-trap and Myc-trap (ChromoTek GmbH, Munich, Germany) or Flag M2 conjugated magnetic beads (Sigma) were added, and the mixture was incubated at 4 °C for 2.5 h. After washing with PBS containing 1% Triton X-100, proteins bound to the beads were eluted by adding 2× electrophoresis sample buffer and analyzed by immunoblotting. The primary antibodies used in the study were as follows: Rabbit and mouse anti-Flag (Sigma Aldrich), mouse anti-Myc (Cell Signaling, Danvers, MA, USA), rabbit anti-Myc (MBL, Tokyo, Japan), mouse anti-GFP (Roche, Basel, Switzerland), mouse anti-GFP (Santa Cruz, Dallas, TX, USA), rabbit anti-NS1.2, anti-NTPase, and anti-NS4. Rabbit anti-NS1.2 was produced in this study immunized rabbit with NS1.2 (1–202), which was purified from bacteria expressed His-tagged NS1.2 (1–202) (AllBio Science Incorporated, Taichung Taiwan). Anti-NTPase and NS4 were kindly provided by Dr. Pey-Jium Chang [33 (link)].
9
GFP Antibody Characterization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oligonucleotides were purchased from Sigma‒Aldrich (Bangalore, India). Mouse anti-GFP was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and mouse anti-His tag antibodies were purchased from Cell Signaling Technology (Denvers, MA). Restriction enzymes, NEB Hi-Fi builder (E2621L) and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA). DNA polymerases were purchased from GeNei (Bangalore, India) and Thermo Fisher Scientific (Waltham, MA).
10
Immunoblot Analysis of Plasmodium Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 2 X105 infected erythrocytes were collected and lysed in 0.5% saponin. The pellet was resuspended in SDS buffer containing 2% β-mercaptoethanol, and separated by SDS-PAGE. After transfer to a nitrocellulose membrane, blots were blocked in 5% milk, and probed with either mouse anti-GFP (1:10,000) or mouse anti- hemagglutinin (HA) epitope (Santa Cruz Biotechnology; 1:10,000 dilution), and subsequently with rabbit or goat anti-mouse IgG conjugated with HRP (secondary 1:1000), and developed on film with SuperSignal West Femto substrate (ThermoFisher Scientific). Plasmodium aldolase was detected using a rabbit anti-aldolase (Abcam) directly conjugated to HRP (1:40,000 dilution).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!