To create MaSINA1-His and MaICE1-GFP constructs, full-length MaSINA1 or MaICE1 was inserted into pEAQ-HT-His and pEAQ-HT-GFP vectors (primers are listed in Supplementary Table 1 ), respectively (Sainsbury et al., 2009 (link); Peyret and Lomonossoff, 2013 (link)), and were introduced into A. tumefaciens strain GV3101, following co-infiltrated into the abaxial side of 4- to 6-wk-old tobacco leaves using a 1-mL needleless syringe. After 36 h of infiltration, 10 μM MG132 (Merck, Cat. No. 474790) was injected into tobacco leaves to prevent protein degradation. After 36 h, tobacco leaves were harvested and the protein was extracted as described by Han et al. (2016) (link) and Ye et al. (2016) (link), as well as the following CoIP assay. A 10 μl volume of anti-GFP antibody (Abcam, Cat. No. ab290) was added to 1 mL of cell lysates. Then, binding was gently shaked at 4°C for 4 h, and 50 μl of protein A agarose beads (Roche, Cat. No. 11134515001) was added. After 3 h of incubation at 4°C, the precipitated samples were washed, separated by SDS-PAGE and then performed western blotting analysis as described above using 4000-fold diluted anti-His antibody (Abcam, Cat. No. ab9108) and anti-GFP antibody (Abcam, Cat. No. ab290) respectively, for CoIP assay, and anti-ubiquitin antibody (Sigma–Aldrich, Cat. No. U119) for examining the ubiquitination of MaICE1.
Anti gfp antibody
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Japan
The Anti-GFP antibody is a laboratory reagent used to detect and visualize green fluorescent protein (GFP) in various experimental systems. It provides a reliable and specific method for identifying and localizing GFP-tagged proteins or cells.
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Lab products found in correlation
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Anti ago2 antibody, by Merck Group (4 mentions)
Pms2 gfp, by Addgene (4 mentions)
Anti flag antibody, by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Lsm 700 confocal microscope, by Zeiss (4 mentions)
Gfp trap beads, by Proteintech (3 mentions)
Bioruptor plus, by Diagenode (3 mentions)
Qiaquick pcr purification kit, by Qiagen (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Dynabeads protein a, by Abcam (3 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti pcna antibody, by Santa Cruz Biotechnology (2 mentions)
Eclipse e600 microscope, by Nikon (2 mentions)
Volocity software v4, by PerkinElmer (2 mentions)
Dmi8 confocal microscope, by Leica (2 mentions)
Fluorsave, by Merck Group (2 mentions)
174 protocols using anti gfp antibody
1
Cloning and Co-Immunoprecipitation of MaSINA1 and MaICE1
2
Cloning and Expression of VmGlu2
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The full-length fragment of VmGlu2 was amplified from the genomic DNA of V. mali and then cloned into plasmid pYF11 using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The construct was verified by sequencing and then transformed into the wild-type strain LXS080601 as described above. Transformants were screened by PCR with corresponding primers outside the cloning sites of the pYF11. Relative transcript levels of VmGlu2 in mycelia grown on PDA and inoculated apple twigs were determined as described above. Transformants were further verified by Western blot with Anti-GFP antibody (Abcam, United Kingdom).
3
ChIP-PCR Assays for Chromatin Analysis
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ChIP‐PCR assays were performed following the method described by Bowler et al. (2004 (link)). Apple calli were crosslinked for 10 min in 1% formaldehyde. Chromatin was isolated and sonicated to shear DNA into 0.2–0.5 kb fragments. After centrifugation at 4 °C for 5 min at 13 000 g , chromatin was retained in the upper aqueous phase. ChIP‐grade protein A/G agarose beads (Thermo Fisher) were used to preclear the chromatin supernatant. An anti‐GFP antibody (Abcam, Cambridge, UK) was added to the supernatant and incubated overnight at 4 °C with gentle agitation. Immune complexes were collected with protein A/G agarose beads after incubation for 1 h at 4 °C with gentle agitation. The beads were centrifuged at 1000 g and then washed five times. Immune complexes were eluted and resuspended. Crosslinking was reversed by overnight incubation at 65 °C in 5 m NaCl. Each ChIP assay was repeated three times and the enriched DNA fragments in each ChIP sample were used as one biological replicate for qPCR. Primers used are listed in Table S5 .
4
MS2-RIP Assay for NNT-AS1 Binding
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For MS2-RIP assay, cell samples were transfected with pcDNA3.1-MS2-NNT-AS1 or pcDNA3.1-MS2 for 48 h. Then, samples were used for RIP assay with anti-GFP antibody (Abcam, Cambridge, MA), using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA). The precipitated RNAs were finally monitored by RT-qPCR. The experiment was repeated at least three times.
5
Chromatin Immunoprecipitation and qRT-PCR
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A 3-g quantity of fresh leaves per sample from seedlings at the rosette stage was harvested, and incubated in 1% formaldehyde in vacuum for 15 min to crosslink protein–RNA. The crosslinking was stopped by adding 125 mM glycine and incubating for 5 min. Leaves were ground and resuspended in lysis buffer (100 mM KCl, 10 mM HEPES, 5 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 0.1% NP-40, 200 U/ml RNasin, 2 mM Ribonucleoside Vanadyl Complexes, 2 mM Phenylmethanesulfonyl fluoride (PMSF) and 10 μl/ml protease inhibitor [PI, Sigma P9599]). The lysed supernatant was sonicated to produce fragments of 300–500 bp, which were then treated with DNase to remove DNA before immunoprecipitation. RNA-IP was then performed using anti-GFP antibody (catalog number ab290, Abcam) coupled with Dynabeads Protein G (catalog number 10003D, Invitrogen). After the preparation was washed five times, the protein on the crosslinked complex was digested by proteinase K, and the RNA was extracted with phenol–chloroform–isoamyl alcohol. Following reverse transcription, qRT-PCR was performed with the UBC30 gene as an internal control.
6
Interaction Analysis of Transcription Regulators
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HEK293T cells were grown in DMEM + 10% FBS and transfected with pCI-Neo-GFP-CTCF and pCI-Neo-HA-BORIS along with pCI-Neo-myc-TAF7L, pReceiver-myc-BRD4 (Genecopoeia), or pReceiver-myc-RFX2 (Genecopoeia). For co-IP analysis, 1.5 mg of total protein was incubated with anti-Myc antibody (Sigma), anti-HA antibody (Roche), or anti-GFP antibody (Abcam ab290) overnight at 4 °C with gentle agitation, followed by incubation with 50 μl of a 1:1 mixture of Protein A and Protein G Dynabeads (Life Technologies) for 1 h at room temperature. The immunoprecipitates were collected using a magnetic rack and washed and analyzed by SDS-PAGE and western blotting.
7
EGFP-TRAIP Protein Interaction Assay
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Cells from stable EGFP/ EGFP-TRAIP HeLa Flp-In T-REx cell lines were fixed with methanol at −20 °C for 10 min followed by a 5 min extraction in 0.3% Triton-X100 in PBS. Cells were then incubated in anti-PCNA antibody (Santa Cruz, PC10; 1:500) and anti-GFP antibody (Abcam, ab6556; 1:500), and in situ proximity ligation was performed using a Duolink Detection Kit in combination with anti-Mouse PLUS and anti-Rabbit MINUS PLA Probes, according to the manufacturer’s instructions (Sigma Aldrich Duolink). Nuclear foci were imaged using a Nikon E600 Eclipse microscope equipped with a 60X oil lens, and images were acquired and analysed using Volocity Software v4.1 (Improvision). The number of nuclear foci/cell was quantified using ImageJ. More than 50 cells were analyzed per experiment per condition.
8
Protein Separation and Western Blot Analysis
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Protein samples were heated at 95°C for about 5 min and then loaded on a 12% polyacrylamide gel [6.8 × 8.6 cm (length × width), 0.75 mm spacer set] for protein separation. After electrophoresis (1 h at 180 V), the gel was stained overnight in the solution of Coomassie Brilliant Blue G-250 with gently shaking. For Western blot assays, a semi-dry electrophoresis transfer unit (Bio-Rad) was used to transfer the proteins from SDS-gel to a 0.45 μm NC membrane (Sigma-Aldrich). Following an overnight-block with phosphate-buffered saline (PBS) made 5% non-fat dry milk, strips from the blot were developed by rabbit polyclonal anti-GFP antibody (Abcam) with 1:1,000 dilutions, next by 1:5,000 goat anti-rabbit HRP conjugated secondary antibody (Sigma-Aldrich). Specific protein bands were visualized after the treatment of the strips with ECL-solution (GE Healthcare).
9
Yeast Protein Sedimentation and Analysis
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The sedimentation assay was adapted from Shiber et al. (50 (link)). Briefly, yeast cultures are normalized to A600 = 1.0 and lysed. Cell lysis was fractioned into whole lysate, supernatant (soluble proteins) and pellet (insoluble proteins). To determine HARS expression, whole cell extracts were separated by SDS-PAGE and transferred to PVDF membrane (Roche Applied Science, Cat. No. 03 010 040 001). Anti-GFP antibody (Abcam, ab32146) was used at ratio of 1:5000. Secondary antibody IRDye® 800CW Goat anti-Rabbit IgG (Li-Cor, 926–32 211) was used at a ratio of 1:20 000 and detected using the Odyssey Classic (Li-Cor). Phostag gels were carried out as described previously (33 (link)) and probed with anti-eIF2α antibody (Invitrogen #AHO082).
10
Co-immunoprecipitation of SUMO2/3 and USP5
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tsA-201 cells or DRG tissues were lysed in a modified RIPA buffer (in mM; 50 Tris, 100 NaCl, 0.2% (v/v) Triton X-100, 0.2% (v/v) NP-40, 10 EDTA + protease inhibitor cocktail, pH 7.5) that was used to co-immunoprecipitate recombinant mCherry-USP5 with Cav3.2-GFP tagged channels with, SUMO2/3 with mCherry-USP5, or native SUMO2/3 with USP5. Lysates were prepared by sonicating samples at 60% pulse for 10 s and by centrifugation at 13,000 rpm for 15 min at 4 °C. Supernatants were transferred to new tubes and solubilized proteins were incubated with 50 μl of Protein G/A beads (Piercenet) and 2 μg of anti-GFP antibody (Abcam) overnight while tumbling at 4 °C. Total inputs were taken from whole cell samples representing 4% of total protein and probed for actin or α-Tubulin. Co-immunoprecipitates were washed twice with (mM) 150 NaCl 50 Tris pH 7.5 buffer, beads were aspirated to dryness. Laemmli buffer was added and samples were incubated at 96 °C for 7 min. Eluted samples were loaded on 7.5% or 10% Tris-glycine gel and resolved using SDS-PAGE. Samples were transferred to 0.45 mm polyvinylidenedifluoride (PDVF) membranes by dry transfer using an Iblot2 machine (Invitrogen).
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