CoIP was performed as previously described (16 ). Briefly, HEK293T cells transiently transfected with the indicated constructs in plates with a diameter of 10 cm were harvested at approximately 48 h posttransfection and centrifuged to remove the cell debris (20,000g, 10 min at 4 °C). Prewashed 20 μl of anti-GFP-Trap-A beads (Chromotek, gta-20) was added and incubated overnight at 4 °C with gentle rotation. The anti-GFP-Trap-A beads were collected by centrifugation at 1000g for 3 min at 4 °C. The beads were washed six times with ice-cold IPB (50 mM Hepes, pH 7.4, 100 mM NaCl, 50 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 1 mM EDTA, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF, one tablet of protease inhibitor cocktail per 10 ml buffer) containing 0.1% Triton X-100. After the last wash, the beads were resuspended in 60 μl IPB buffer, and the immunoprecipitated proteins were eluted in 3× loading buffer and analyzed by Western blotting after boiling for 5 min at 100 °C.
Anti gfp trap a beads
Manufactured by Proteintech
Sourced in Germany
Anti-GFP-Trap-A beads are sepharose beads coated with a recombinant GFP-binding protein. They can be used to immunoprecipitate and isolate GFP-tagged proteins and their associated complexes from cell lysates.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescent reagent, by GE Healthcare (1 mentions)
Hrp 66005, by Proteintech (1 mentions)
Pegfp c1 expression vector, by Takara Bio (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Roche (1 mentions)
Q exactive series mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Rippa buffer, by Merck Group (1 mentions)
4 protocols using anti gfp trap a beads
1
Co-Immunoprecipitation (CoIP) Protocol
2
Immunoprecipitation of GFP-tagged Proteins
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Xenopus laevis oocytes were processed as described previously; anti-GFP-Trap-A beads (ChromoTek, Germany) were used for immunoprecipitation [22 (link)]. Laemmli buffer was used to recover the immunoprecipitates, and eluates were analyzed separately using the following primary antibodies: mouse monoclonal anti-GFP antibody (Proteintech # 66002-1-Ig, 1:10000), HRP-conjugated anti-His antibody (Proteintech # HRP-66005). HRP conjugated anti-mouse antibody was used as the secondary antibody (1:10000; SA00001-1). Chemiluminescent reagent (GE healthcare, IL, USA) was used for detection.
3
LRGUK-1 Interaction with HOOK2 in HEK293 Cells
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Full length mouse Lrguk-1 was amplified from wild type C57BL/6J testis cDNA using Lrguk-Fw: 5’-ATATAAAGATCTGCGGCCTTCGAGCGAAAT-3’ and Lrguk-Rev: 5’-ATATAAGGGCCCCTATCGCGGCCGTGCGGGAT-3’ primers than cloned into the pEGFP-C1 expression vector (Clontech, Gen Bank Accession number U55763). The LRGUK-1/pEGFP-C1 construct was transfected into HEK293 cells using Lipofectamine 3000 Reagent (Life Technologies, Cat. No. L3000008). Co-immunoprecipitation was carried out using anti-GFP-Trap-A beads as per the manufacturer’s instructions (Chromotek, Cat. No. gta-100). Empty pEGFP-C1 vector was used as a negative control. The presence of LRGUK-GFP and endogenous HOOK2 proteins were determined using anti-GFP (Roche, Cat. No. 11814460001 and HOOK2 antibodies (GeneTex, Cat. No. GTX115898, corresponding with amino acids 197–380 of HOOK2).
4
Immunoprecipitation and Mass Spectrometry Analysis of GmNFYA-GFP
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For immunoprecipitation followed by mass spectrometry, soybean transgenic hairy roots harbouring 35S:GmNFYA‐GFP (NFYA‐GFP) were generated and GFP‐overexpressing hairy roots were used as control. Three gram of transgenic hairy roots were harvested and total proteins were extracted in Rippa buffer (Sigma). Anti‐GFP Trap‐A beads (Chromotek) was used for IP. After IP, the proteins were separated by 10% SDSS‐PAGE. The stained protein band was cut and in‐gel digestion was performed by Novogene (Beijing, China). Mass spectrometry measurements were performed on a Q ExactiveTM series mass spectrometer (Thermo Fisher). The resulting spectra were searched against UNIPROT protein database, using soybean as a target organism. Gene ontology (GO) and InterPro (IPR) functional analysis were conducted using the interproscan programme against the non‐redundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSite, PANTHER), and the databases of COG (Clusters of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used to analyse the protein family and pathway. Proteins uniquely identified in NFYA‐GFP sample but not in control sample were taken into account.
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