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.
Na931v
Manufactured by Merck Group
The NA931v is a laboratory equipment product from Merck Group. It is designed to perform a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation. Further information is not available.
Automatically generated - may contain errors
Lab products found in correlation
Na934v, by Merck Group (4 mentions)
Ab290, by Abcam (3 mentions)
Rfp antibody 6g6, by Proteintech (3 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Gerpn1236, by Merck Group (2 mentions)
Gfp trap agarose beads, by Proteintech (2 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Rabbit anti caspase 3 mab, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Rab11a, by Thermo Fisher Scientific (1 mentions)
7 protocols using na931v
1
Protein-Protein Interaction Mapping using Pull-Down Assays
2
In Vitro Pull-Down Assay of DA2 by UBP12/13
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For the in vitro pull-down assays of DA2 by UBP12 or UBP13, around 2 μg of GST-UBP12, GST-UBP13, or GST proteins was incubated with 5 μg of HIS-MBP-DA2 protein in 1 ml of pull-down buffer [10% glycerol, 1% Triton X-100, 150 mM NaCl, 50 mM Hepes (pH 7.5), and 1× cOmplete protease inhibitor cocktail (Roche, 11697498001)]. These reactions were incubated with 15 μl of GST beads (glutathione Sepharose 4B agarose) while gently shaking at 4°C for 1 hour. The beads were washed six times, and the proteins were eluted with 35 μl of GST elution buffer [50 mM tris-HCl (pH 8.0), 10 mM reduced glutathione, and 10% glycerol] for 30 min at 4°C. The eluted samples were heated for 10 min at 95°C after the addition of 5× sample buffer. The immunoprecipitated proteins were separated by SDS–polyacrylamide gel electrophoresis and detected with anti-GST HRP conjugate (1:3000; Sigma-Aldrich, GERPN1236, RRID:AB_2827942) and MBP-tagged proteins with anti-MBP monoclonal antibody (1:10,000; NEB, E8030S, RRID:AB_1559728) and subsequently with a secondary mouse IgG HRP-linked antibody (1:5000; Sigma-Aldrich, NA931v, RRID:AB_2827944). 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
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.
5
Western Blot Analysis of Autophagy and Apoptosis Markers
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Proteins were separated by polyacrylamide gel electrophoresis (15% or 7.5%) with sodium dodecyl sulfate (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked by 5% milk in TBS-Tween (Tris-Buffered Saline 0.05% Tween 20) for 1 h, and then incubated with rabbit anti-LC3 pAb (NB100-2331 Novus Biologicals, Centennial, CO, USA), rabbit anti-SQSTM1 mAb (8025, Cell Signaling Technology, Boston, MA, USA), rabbit anti-PARP monoclonal antibody mAb (9542, Cell Signaling Technology), or rabbit anti-caspase 3 mAb (9661, Cell Signaling Technology), all of them diluted 1:1000 in TBS-Tween. Membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (A1949, Sigma-Aldrich). Mouse anti-β-actin mAb (A5316, Sigma-Aldrich) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (NA931V, Sigma-Aldrich) were used to have a loading control. Immunoreactivity was assessed through the development of a chemiluminescence reaction using an ECL detection system (Amersham, Buckinghamshire, UK). Densitometric analysis was performed by Mac OS X (Apple Computer International, Cupertino, CA, USA), using NIH Image 1.62 software.
6
Western Blot Analysis of Cell Lysates
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Cells were lysed in RIPA buffer (50mM Tris pH 7.4, 150mM NaCl, 0.1% SDS, 0.5% deoxy cholate, 1% Triton X-100) supplemented with protease inhibitors (Sigma), quantified, and protein samples were separated on a 10% SDS-PAGE gel. Western blot analysis was performed following the transfer of proteins onto a nitrocellulose membrane. Antibodies used were NP (#4862, BEI resources), Ku (#2882, Sigma), Rab11a (#715300, Invitrogen), and goat α-mouse or goat α-rabbit secondary antibodies conjugated to horseradish peroxidase (#NA934V and NA931V, Sigma).
7
In Vitro Pull-Down Assay of DA2 by UBP12/13
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For the in vitro pull-down assays of DA2 by UBP12 or UBP13, around 2 μg of GST-UBP12, GST-UBP13, or GST proteins was incubated with 5 μg of HIS-MBP-DA2 protein in 1 ml of pull-down buffer [10% glycerol, 1% Triton X-100, 150 mM NaCl, 50 mM Hepes (pH 7.5), and 1× cOmplete protease inhibitor cocktail (Roche, 11697498001)]. These reactions were incubated with 15 μl of GST beads (glutathione Sepharose 4B agarose) while gently shaking at 4°C for 1 hour. The beads were washed six times, and the proteins were eluted with 35 μl of GST elution buffer [50 mM tris-HCl (pH 8.0), 10 mM reduced glutathione, and 10% glycerol] for 30 min at 4°C. The eluted samples were heated for 10 min at 95°C after the addition of 5× sample buffer. The immunoprecipitated proteins were separated by SDS–polyacrylamide gel electrophoresis and detected with anti-GST HRP conjugate (1:3000; Sigma-Aldrich, GERPN1236, RRID:AB_2827942) and MBP-tagged proteins with anti-MBP monoclonal antibody (1:10,000; NEB, E8030S, RRID:AB_1559728) and subsequently with a secondary mouse IgG HRP-linked antibody (1:5000; Sigma-Aldrich, NA931v, RRID:AB_2827944). The protein blots were visualized as described in the previous section.
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