The in vitro pull-down experiments were performed as described before (Dong et al., 2017 (link)). As recombinant bait proteins, we used equal quantities of recombinant GST (Prospec, ENZ-393) and full-length GST-UBP12 or GST-UBP13. For the in vivo pull-down, full expression constructs of 35S::RFP-DA1, 35S::RFP-DAR1, 35S::RFP-DAR2, 35S::GFP-UBP12 and 35S::GFP were amplified by PCR and cloned into Golden Gate modules (pGGA000, pGGB000) using the Gibson assembly method (NEB, E5510S). Existing bsaI sites in the Gateway p35S and DAR1 coding sequence were mutated by site-directed mutagenesis using DpnI. Subsequently, these constructs were cloned as interaction pairs on a Golden Gate vector to ensure the co-expression of the constructs in each cell. Arabidopsis cell suspension cultures were transformed as described before (Van Leene et al., 2011 (link)). Proteins were extracted and purified as described above and the purified fraction was subjected to Western blot. After blocking, GFP-tagged proteins were detected with anti-GFP (Abcam, ab290, RRID:AB_303395 ) and RFP-tagged proteins with anti-RFP antibody (Chromotek, RFP antibody [6G6], RRID:AB_2631395 ) and subsequently with a secondary Rabbit IgG HRP-linked antibody (Sigma-Aldrich, NA934v, RRID:AB_2722659 ) or secondary Mouse IgG HRP-linked antibody (Sigma-Aldrich, NA931v, RRID:AB_2827944 ), respectively.
Rfp antibody 6g6
Manufactured by Proteintech
The RFP antibody [6G6] is a mouse monoclonal antibody that specifically recognizes the red fluorescent protein (RFP) tag. This antibody can be used for the detection and immunoprecipitation of RFP-tagged proteins in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab290, by Abcam (3 mentions)
Na934v, by Merck Group (3 mentions)
Na931v, by Merck Group (3 mentions)
Gfp trap agarose beads, by Proteintech (2 mentions)
Ab6160, by Abcam (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Bio-Rad (2 mentions)
α flag, by Merck Group (2 mentions)
α tubulin, by Abcam (2 mentions)
α gapdh, by Abcam (2 mentions)
α rat, by Merck Group (2 mentions)
α mcherry, by Proteintech (2 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Goat anti mouse igg1 cy5, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit cy5, by Jackson ImmunoResearch (1 mentions)
Iba1 antibody 019 19741, by Fujifilm (1 mentions)
Alexa fluor 647 donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Gfp trap, by Proteintech (1 mentions)
7 protocols using rfp antibody 6g6
1
Protein-Protein Interaction Mapping using Pull-Down Assays
2
Detecting GFP and mCherry Proteins
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Vectors that simultaneously carried constructs of free GFP, GFP-UBP12, or GFP-UBP13 with mCherry-DA2 were transformed in cell suspension cultures, and the protein extraction was performed as described by Van Leene et al. (103 (link)). Proteins were extracted and purified with GFP-Trap Agarose beads (ChromoTek, RRID:AB_2631357), and the purified fractions were subjected to Western blot. After blocking, GFP-tagged proteins were detected with anti-GFP antibody (1:1000; Abcam, ab290, RRID:AB_303395) and mCherry-tagged proteins with anti-RFP antibody (1:1000; ChromoTek, RFP antibody [6G6], RRID:AB_2631395) and subsequently with a secondary rabbit IgG HRP-linked antibody (1:2000; Sigma-Aldrich, NA934v, RRID:AB_2722659) or secondary mouse IgG HRP-linked antibody (1:5000; Sigma-Aldrich, NA931v, RRID:AB_2827944), respectively. The protein blots were visualized as described in the previous section.
3
Detecting GFP and mCherry Proteins
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Vectors that simultaneously carried constructs of free GFP, GFP-UBP12, or GFP-UBP13 with mCherry-DA2 were transformed in cell suspension cultures, and the protein extraction was performed as described by Van Leene et al. (103 (link)). Proteins were extracted and purified with GFP-Trap Agarose beads (ChromoTek, RRID:AB_2631357), and the purified fractions were subjected to Western blot. After blocking, GFP-tagged proteins were detected with anti-GFP antibody (1:1000; Abcam, ab290, RRID:AB_303395) and mCherry-tagged proteins with anti-RFP antibody (1:1000; ChromoTek, RFP antibody [6G6], RRID:AB_2631395) and subsequently with a secondary rabbit IgG HRP-linked antibody (1:2000; Sigma-Aldrich, NA934v, RRID:AB_2722659) or secondary mouse IgG HRP-linked antibody (1:5000; Sigma-Aldrich, NA931v, RRID:AB_2827944), respectively. The protein blots were visualized as described in the previous section.
4
Western Blot Analysis of C. elegans Proteins
5
Western Blot Analysis of C. elegans Proteins
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Protein extracts, prepared as above, were resolved on precast NuPAGE™ Novex 4-12% Bis-Tris gels (Invitrogen NP0321BOX). The proteins were transferred to a nylon membrane with the semidry transfer Thermo Scientific™ Pierce™ Power System using the Pre-Programmed Method for High MW protein. The primary antibody used were α-PRG-1 antibody6 (link) (a gift from the Mello lab), α-CSR-1 antibody61 (link), α-PGL-162 (link) and α-DEPS-138 (link) (a gift from the Strome lab), α-FLAG (F3165, Sigma), α-GAPDH (Ab125247, Abcam), α-mCherry (RFP antibody [6G6], Chromotek), α-tubulin (Ab6160, Abcam), and the secondary antibody used were α-rabbit (31460, Pierce), α-mouse (31430, Pierce), or α-Rat (A9037, Sigma) HPR antibodies. The SuperSignal™ West Pico PLUS Chemiluminescent Substrate was used to detect the signal with the ChemiDoc™ MP imaging system (Biorad).
6
Immunofluorescence Antibody Protocol
7
Extracting Proteins from N. benthamiana
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Proteins were extracted from transiently transformed N. benthamiana leaves using 1 ml lysis buffer (20 mM HEPES, pH 7.5, 40 mM KCl, 1 mM EDTA, 0.1% [v/v] Triton X-100, 1× protease inhibitor cocktail, 1 mM phenylmethylsulfonyl fluoride, and 10% [v/v] glycerol) (Ganguly et al., 2020) . Homogenate from 500 mg of plant material was centrifuged twice at 4°C at 15000 g for 10 min, and 900 µl of the supernatant was incubated with 20 µl GFP-Traps (Chromotek) overnight at 4°C on a rocking shaker. Beads were equilibrated following manufacturer's manual using lysis buffer, without Triton-X. The next day, beads were washed four times and were boiled in 80 µl 2x Laemmli Buffer. For each sample, 40 µl were loaded on a SDS gel and blotted afterwards for 1 h. For protein detection, 1:1000 dilution of the GFP antibody 3H9 (Chromotek) or the RFP antibody 6G6 (Chromotek) was used. Anti-mouse (Sigma A9044 1:20000) and anti-rat (Thermo 31470 1:3000) secondary antibodies conjugated to horseradish peroxidase were used to detect RFP and GFP signals, respectively. Western blot images were acquired with a FluorChem system, using 1:1 mixture of Amersham ECL-Prime and ECL-Select as chemiluminescent detection reagents.
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