Nuclear extracts were prepared as described earlier [31 ]. One milliliter nuclear extract (5–10 mg of protein) was incubated with 20 µL of GFP nano‐trap beads (Chromotek), in the presence of 50 µg·µL−1 ethidium bromide. After 1.5‐h incubation at 4 °C, beads were washed thoroughly with a stringent washing buffer (2× buffer Cextra (1 m NaCl, 20 mm HEPES/KOH pH 7.9, 20% glycerol, 2 mm MgCl2, 0.2 mm EDTA, 1.0% NP40, 0.5 mm DTT, complete protease inhibitors (Roche)), 2× PBS with 1.0% NP40, and 2× PBS). Subsequently, proteins were cross‐linked using 2 mm BS3 or 43.4 mm DMTMM + 47.6 mm AHD in borate‐buffered saline at 37 °C for 1 h [14 ], which was quenched with 100 mm Tris/HCl pH 8.0 for 10 min. Proteins were subjected to on‐bead digestion using trypsin or chymotrypsin (0.25 µg protease in 2 m urea, 100 mm Tris/HCl pH 8.5, 10 mm DTT) prior to STAGE tipping [32 ].
Gfp nanotrap beads
Manufactured by Proteintech
Sourced in Germany
GFP-nanotrap beads are a type of affinity matrix designed for the purification of Green Fluorescent Protein (GFP) and GFP-fusion proteins. These beads are coated with a highly specific nanobody that binds to GFP with high affinity, allowing for efficient capture and isolation of GFP-tagged proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Mini complete protease inhibitor tablet, by Proteintech (2 mentions)
Orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 precast gels, by Thermo Fisher Scientific (1 mentions)
Silac reagents, by Thermo Fisher Scientific (1 mentions)
Silac dmem, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
Complete mini protease inhibitor tablet, by Roche (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Thermo orbitrap exploris 480, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (1 mentions)
11 protocols using gfp nanotrap beads
1
Protocol for Nuclear Protein Isolation and Crosslinking
2
Affinity Purification of GFP-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared from the cells described above in 0.5 ml lysis buffer (50 mM Kpi pH7, 250 mM NaCl, 1% Triton X100, 0.5% deoxycholate, 1 mM MgCl2, 5 mM DTT and protease inhibitors) with 0.5 mm beads in a Fastprep machine. After bead-beating, the cells were incubated on ice for 15 min, and subsequently centrifuged at 20,000 rcf for 15 min to clear the lysates of beads and unbroken cells. The cell lysates were diluted with lysis buffer to 1 ml and 10 µl of GFP-nanotrap beads (Chromotek) were added. After 1.5 hr of incubation, the beads were washed six times with wash buffer (50 mM Kpi pH7, 250 mM NaCl, 0.1% SDS, 0.05% Triton X100, 0.025% Deoxycholate, protease inhibitors). Bound proteins were subsequently eluted from the beads by adding 20 µl of 10% SDS to the beads and 10 min of incubation at 95°C. As a negative control a cell lysate from BY4741, not expressing any GFP-tagged nucleoporin, was treated as above. As positive controls BY4741 was spiked with 2 µg of purified ID-GFP, or 2 µg of ID-GFP which was first in vitro oxidized with 1 mM CuSO4 and 4 mM H2O2 for 15 min at RT.
3
Quantitative Proteomic Analysis of SNX21
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SILAC reagents were purchased from Thermo Fisher with the exception of FCS and SILAC DMEM from Sigma. RPE1 cells virally expressing selected plasmids were seeded in six-well plates in SILAC labelling medium supplemented with dialysed FCS and cultured over a minimum of six passages to achieve full labelling with respective isotopes and a minimum of two confluent 20 cm dishes for generation of lysates. GFP-expressing control cells were grown in unlabelled medium with standard arginine and lysine (R0K0) and GFP-SNX21-expressing cells were grown in medium supplemented with ‘medium’-mass isotopes [13C6]-arginine and 4,4,5,5-D4-Lysine (R6K4). Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer (Steinberg et al., 2012 (link); Steinberg et al., 2013 (link); McGough et al., 2014a (link),b (link); McMillan et al., 2016 (link)).
4
GFP-based and Antibody-mediated Immunoprecipitation Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For GFP-based immunoprecipitations (IPs), cells were lysed in 10 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA and 0.5% NP-40 complemented with protease and phosphatase inhibitors. The supernatant was collected and incubated with GFP nanotrap beads (Chromotek) for 3 h at 4 °C. To elute EGFP–KIND2 and associated proteins, the beads were washed with 10 mM Tris-HCl, 150 mM NaCl and 0.5 mM EDTA three times and boiled in 2× Laemmli buffer at 95 °C for 10 min. The eluted fraction was then run on SDS–PAGE followed by WB or MS analysis.
For antibody-based IPs, cells were lysed as described above, incubated with the respective antibody overnight at 4 °C, and then incubated with protein A/G agarose beads (Santa Cruz sc-2003) for 3 h at 4 °C. The beads were collected, washed and eluted as mentioned above.
For antibody-based IPs, cells were lysed as described above, incubated with the respective antibody overnight at 4 °C, and then incubated with protein A/G agarose beads (Santa Cruz sc-2003) for 3 h at 4 °C. The beads were collected, washed and eluted as mentioned above.
5
GFP-nanotrap and SNX21 Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For GFP-nanotrap, HEK293-T cells were transfected with respective GFP constructs as described above, lysed in immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 0.5% NP40, 1 mM PMSF, 200 µM Na3VO4 and a Roche mini complete protease inhibitor tablet) and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C, washed three times in wash buffer (50 mM Tris-HCl, pH 7.4, 0.2% NP40), prior to separation by SDS-PAGE and western blotting. For the endogenous immunoprecipitations; HEK293-T cells were lysed in immunoprecipitation buffer (50 mM Tris-HCL, pH 7.4, 0.5% NP40, 1 mM PMSF, 200 µM Na3VO4 and a Roche mini complete protease inhibitor tablet). Cleared lysates were separated into two fractions prior to the addition of 2 µg of either anti-SNX21 or rabbit IgG control antibody and incubated at 4°C for 2 h. Protein-G agarose beads (Pierce) were then washed three times in lysis buffer and added to the respective lysates prior to a further incubation at 4°C for 1 h. Beads were washed three times in wash buffer (50 mM Tris-HCl, pH 7.4, 0.2% NP40), prior to separation by SDS-PAGE and western blotting.
6
SILAC-Based GFP Interactome Profiling
7
Affinity Purification of Chromatin Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear extracts were prepared as described earlier [31 ]. The affinity purifications are performed in triplicate, and so are the negative control purifications. For CHD4, the negative control purification consists of a GFP affinity purification on WT nuclear extract. For the CDK2AP2 purifications, blocked agarose beads were used as a negative control on the same nuclear extract. One mg of nuclear extract was incubated with 7.5 µL of GFP nano‐trap beads (Chromotek, Planegg‐Martinsried, Germany), in the presence of 50 µg·µL−1 ethidium bromide. After 1.5‐h incubation at 4 °C, beads were washed thoroughly (2× buffer C (1 m NaCl, 20 mm HEPES/KOH pH 7.9, 20% glycerol, 2 mm MgCl2, 0.2 mm EDTA, 0.25% NP40, 0.5 mm DTT, complete protease inhibitors (Roche, Basel, Switzerland)), 2× PBS with 0.25% NP40, and 2× PBS). Purified proteins were subjected to on‐bead digestion using trypsin (0.25 µg protease in 2 m urea, 100 mm Tris/HCl pH 8.5, 10 mm DTT) prior to STAGE tipping [32 ].
8
Affinity Purification and Western Blotting of GFP-Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were grown to 85% confluence in 15 cm dishes prior to transfection with 10 µg of plasmid DNA using PEI (Sigma-Aldrich), and incubated for 48 h prior to immunoprecipitation using GFP nanotrap beads (Chromotek)40 (link)–43 (link). Western blots were performed using standard procedures. Detection was carried out on a Licor Odyssey Infrared scanning system using fluorescently labelled secondary antibodies. Uncropped versions of the blots are shown in Supplementary Fig. 4 .
9
GFP Nano-trap Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 20 µl of resuspended GFP nano-trap beads (Chromotek, Planegg-Martinsried, Germany) per sample was added to 500 µl of ice-cold dilution buffer (10 mM Tris-HCl pH 7.4, 50 mM NaCl, 0.5 mM EDTA, freshly added 1 mM PMSF and 1× EDTA-free Protease Inhibitor Cocktail), spun down at 2700 g for 2 min at 4°C, and the supernatant discarded. This wash was repeated two more times. The cell lysate supernatants were added to the equilibrated beads and incubated on a rotor at 4°C for 2 h.
10
GFP-mediated Chromatin Pull-down Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear extraction was performed as previously reported [46 (link)].
GFP pull-down of GFP/CBX2 HeLa and wt HeLa nuclear extracts was performed in triplicate using GFP Nano-Trap beads (Chromotek). For each pull-down, 1 mg of nuclear extract was incubated with 15 µL beads in incubation buffer (300 mM NaCl, 0.15% NP-40, 0.5 mM DDT, 20 mM HEPES-KOH pH 7.9) containing ethidium bromide at a final concentration of 50 mg/ml to prevent indirect DNA-mediated interactions. Beads were then washed twice with incubation buffer containing 0.5% NP-40, twice with PBS containing 0.5% NP-40, and finally twice with PBS alone.
GFP pull-down of GFP/CBX2 HeLa and wt HeLa nuclear extracts was performed in triplicate using GFP Nano-Trap beads (Chromotek). For each pull-down, 1 mg of nuclear extract was incubated with 15 µL beads in incubation buffer (300 mM NaCl, 0.15% NP-40, 0.5 mM DDT, 20 mM HEPES-KOH pH 7.9) containing ethidium bromide at a final concentration of 50 mg/ml to prevent indirect DNA-mediated interactions. Beads were then washed twice with incubation buffer containing 0.5% NP-40, twice with PBS containing 0.5% NP-40, and finally twice with PBS alone.
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!