To confirm the interaction between two RAS proteins and Ram1 in vivo, the coding regions for RAS1 or RAS2, respectively, were cloned into pKNRG, and the resulting constructs were pKNRG‐RAS1 and pKNRG‐RAS2. The coding region for Ram1 was cloned into pKNFLAG to gain the pKNFLAG‐Ram1. The pKNRG‐RAS1/pKNFLAG‐Ram1 and pKNRG‐RAS2/pKNFLAG‐Ram1 were co‐transformed into protoplasts of strain P131. To perform the Co‐IP assay, total proteins were extracted from the resulting transformants and then incubated with the anti‐FLAG M2 affinity resins (Sigma‐Aldrich, St. Louis, MO, USA). Proteins eluted from the M2 resins were analysed by western blot with the anti‐FLAG and anti‐GFP antibodies (Abmart, Xuhui, Shanghai, China).
Anti flag m2 affinity resin
Manufactured by Merck Group
Sourced in United States
The Anti-FLAG M2 affinity resin is a chromatography resin designed for the purification of proteins that have been engineered to contain a FLAG tag. The resin consists of an agarose support matrix with immobilized anti-FLAG M2 monoclonal antibody, which specifically binds to the FLAG peptide sequence. This resin can be used to capture and purify FLAG-tagged proteins from cell lysates, culture supernatants, or other sample types.
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Lab products found in correlation
Flag peptide, by Merck Group (14 mentions)
3xflag peptide, by Merck Group (8 mentions)
Anti flag, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Protein g sepharose bead, by GE Healthcare (3 mentions)
Dh10bac, by Thermo Fisher Scientific (3 mentions)
A8592, by Merck Group (2 mentions)
Synthetic 3 flag peptide, by Apexbio (2 mentions)
Flag peptide, by GenScript (2 mentions)
Amylose resin, by New England Biolabs (2 mentions)
Tpx2 proteins, by Sino Biological (2 mentions)
Aurora a proteins, by Sino Biological (2 mentions)
Benzonase, by Merck Group (2 mentions)
Protein g agarose resin, by Merck Group (2 mentions)
Protein g agarose resin, by Roche (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Storm 840, by GE Healthcare (2 mentions)
Amicon ultra 100k, by Merck Group (2 mentions)
Fast protease inhibitor, by Merck Group (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
80 protocols using anti flag m2 affinity resin
1
Confirming RAS-Ram1 Protein Interaction
2
Purification and Binding of Recombinant USP7 Protein
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Recombinant 3xFLAG-tagged USP7 proteins were expressed in Sf9 insect cells. These insect cells were collected and suspended in lysis buffer (20 mM Tris-HCl [pH 8.0], 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1% NP-40, 1 mM DTT, 5 µg/ml leupeptin, 2 µg/ml aprotinin, 20 µg/ml trypsin inhibitor, and 100 µg/ml phenylmethylsulfonyl fluoride [PMSF]), followed by incubation on ice for 10 min. Soluble fractions were isolated after centrifugation of the lysate at 15,000× g for 15 min at 4°C. 2 ml of the soluble lysate was incubated with 30 µl of anti-FLAG M2 affinity resins (Sigma-Aldrich) for 2 hr at 4°C. The protein-bound beads were washed five times with wash buffer (20 mM Tris-HCl [pH 8.0], 100 mM KCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 1 mM DTT, 5 µg/ml leupeptin, 2 µg/ml aprotinin, 20 µg/ml trypsin inhibitor, and 100 µg/ml PMSF) and stored in PBS at 4°C. 10 µl of protein-bound FLAG beads were coupled with 100 µl of Xenopus egg extracts diluted fivefold by CPB containing 2% sucrose and incubated for 2 hr at 4°C. The beads were washed three times by CPB containing 2% sucrose and 0.1% Triton X-100, followed by resuspension by 10 µl of 2× Laemmli sample buffer and 20 µl of 1× Laemmli sample buffer.
3
In vivo Interactions of MoCapA, MoCapB, and MoAct1
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To confirm the interactions of MoCapA, MoCapB, and MoAct1 in vivo, the cDNA of MoACT1 was cloned into pKNFLAG, the cDNA of MoCAPA was cloned into pKNFLAG or pKNRG, and the cDNA of MoCAPB was cloned into pKNRG. The MoCAPA-3xFLAG and MoCAPB-GFP fusion constructs were introduced by co-transformation into protoplasts of the wild-type strain. Total proteins were isolated from transformants expressing both MoCAPA-3xFLAG and MoCAPB-GFP and incubated with anti-FLAG M2 affinity resins (Sigma Aldrich). Proteins bound to M2 resins were eluted after a series of washing steps as manufacture’s instruction. Western blots of total proteins and elution from M2 resins were detected with anti-FLAG (Sigma Aldrich) and anti-GFP (Abmart) antibodies using the ECL Supersignal system (Pierce, Rockford, IL). Similar methods were used to detect interactions of MoACT1-3xFLAG/ MoCAPA-GFP and MoACT1-3xFLAG/ MoCAPB-GFP.
4
Protein Interactions Validated by Co-IP Assays
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Co-immunoprecipitation (Co-IP) assays were used to confirm the protein interactions in vivo. ChPhb1 cDNA was cloned into pNeo3300-FLAG, while ChPhb2 cDNA was cloned into pNeo3300-GFP. The resulting vectors (ChPhb1–3×FLAG and ChPhb2-GFP) were co-transformed into the wild-type strain. Total proteins were isolated from transformants expressing both ChPhb1–3×FLAG and ChPhb2-GFP, and incubated with anti-FLAG M2 affinity resins (Sigma Aldrich). Proteins that bound to the M2 resins were eluted after a series of washing steps following the manufacturer’s instructions. Immunoblot analysis was performed with anti-FLAG (1:2000 dilution; Sigma Aldrich, USA) and anti-GFP (1:10000 dilution; TransGen Biotech, China) primary antibodies. Immunoblot signals were detected using the ChemiDoc XRS + system (Bio-Rad). Interactions of ChPhb1–3 × FLAG/ChPhb2-GFP, ChPhb1–3 × FLAG/ChATG24-GFP and ChATG24-3 × FLAG/ChPhb2-GFP were also measured based on the above methods. Similar methods were used to test the deletion strain ΔChPhb1 expressing ChATG24-3 × FLAG with ChPhb2-GFP fusion constructs, and deletion strain ΔChPhb2 expressing ChPhb1–3 × FLAG with ChATG24-GFP fusion constructs.
5
Investigating Ebg1 and EF1α Interaction
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To confirm the interactions of Ebg1 and EF1α in vivo, the genomic sequence of EBG1 was cloned into pGTN containing a GFP tag under its native promoter, while the genomic sequence of EF1α was cloned into pTH3Flag under its native promoter (Supplementary Table 3 ). The resulting constructs pGTN-EBG1 and pTH3Flag-EF1α were co-transformed into protoplasts of the wild-type strain P131. Transformants were screened on CM plates containing both neomycin (400 μg/ml, Ameresco) and hygromycin B (250 μg/ml, Roche). Total proteins from culture filtrates (CM) of the transformant expressing both Ebg1-GFP and EF1α-3Flag were incubated, respectively, with anti-GFP affinity resins (Chromotek, gta-20) and anti-Flag M2 affinity resins (Sigma-Aldrich, F2426). Proteins bound to resins were eluted after a series of washing steps following the manufacturer’s instruction. The total proteins and elution from resins were detected by western blotting.
For western blotting detecting the presence of proteins, the following antibodes were used, anti-Actin (ABclonal, AC009, 1:2500), anti-Flag (Sigma, A8592, 1:5000), anti-HA (Sigma, H3663, 1:5000), anti-His (Abmart, 10E2, 1:5000), anti-p44/42 (Cell signaling, 9101, 1:2500), anti-GFP (Abclonal, AE012, 1:5000), anti-GST (EASYBIO, BE7012, 1:5000).
For western blotting detecting the presence of proteins, the following antibodes were used, anti-Actin (ABclonal, AC009, 1:2500), anti-Flag (Sigma, A8592, 1:5000), anti-HA (Sigma, H3663, 1:5000), anti-His (Abmart, 10E2, 1:5000), anti-p44/42 (Cell signaling, 9101, 1:2500), anti-GFP (Abclonal, AE012, 1:5000), anti-GST (EASYBIO, BE7012, 1:5000).
6
Whole-Cell Lysate Preparation and FLAG-ADD1 Immunoprecipitation
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To prepare whole‐cell lysates, cells were lysed with 1% NP‐40 lysis buffer (1% NP‐40, 20 mM Tris–HCl pH 8.0, 137 mM NaCl, 10% glycerol, and 1 mM Na3VO4) containing protease inhibitor cocktail (Roche). To precipitate FLAG‐ADD1, the cells were lysed in TBS buffer (1% Triton X‐100, 50 mM Tris–HCl pH 7.4, and 150 mM NaCl) containing protease inhibitors and subjected to centrifugation at 19,200 × g at 4°C for 10 min. The cell lysates were incubated with anti‐FLAG M2 affinity resins (Sigma‐Aldrich) for 16 h according to the manufacturer's instructions. FLAG‐ADD1 proteins were eluted from the resins with 100 μM 3× FLAG peptide (Sigma‐Aldrich), boiled for 3 min in SDS sample buffer, subjected to SDS–polyacrylamide gel electrophoresis, and transferred to nitrocellulose (Schleicher and Schuell). Immunoblotting was performed with the indicated antibodies using the Western Chemiluminescent HRP Substrate (Immobilon; EMD Millipore) for detection. Chemiluminescent signals were detected by a luminescence imaging system (Fuji LAS‐4000 mini).
7
FLAG-Affinity Purification Protocol
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Cells were lysed in lysis buffer for 20 min with gentle rocking at 4 °C. Lysates were cleared by centrifugation and then filtered through 0.45 μm spin filters (Millipore) to further remove cell debris, and the resulting material was subjected to IP with 50 μL anti-FLAG M2 affinity resin (Sigma) overnight at 4 °C. Resin-containing immune complexes was washed with ice-cold lysis buffer followed by Tris buffered saline (TBS) washes. Proteins were eluted with two-50 μL 150 μg/mL 3× Flag-peptide (Sigma) in TBS for 30 min, and the elution was pooled for a final volume of 100 μL. Proteins in each elution were precipitated with cold acetone. Three biological replicates were carried out.
8
Purification of Halo-tagged MYA2 and Actin
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Protein purification was performed as previously described with some modifications35 (link). Cells were harvested and washed with 150 mM NaCl, 1 mM EGTA, and 10 mM HEPES, pH 7.4. The pelleted cells were suspended with 2 vol/g cells of buffer A (30 mM HEPES, pH 7.4, 200 mM KCl, 5 mM MgCl2, 4 mM ATP, 1 mM EGTA, 1 mM DTT, and a mixture of protease inhibitors). Then, 2 vol/g cells of buffer A containing 2% Nonidet P-40 was added and mixed. After incubation on ice for 10 min, the lysate was centrifuged at 228,000×g for 30 min. The supernatant was mixed with 0.3 mL of anti-FLAG M2 affinity resin (Sigma-Aldrich, St. Louis, MO, USA) in a 50 mL tube on a rotating wheel for one hour at 4 °C. The resin suspension was then loaded on a column and washed with 30 mL of buffer A containing 1 µM mouse calmodulin. Halo-tag-fused MYA2 was biotinylated by incubation with buffer A containing 10 µM of HaloTag PEG-Biotin Ligand (Promega) in the column for 10 min. MYA2 and Halo-tag-fused MYA2 was eluted with buffer A containing 0.2 mg/mL of 3× FLAG peptide (Sigma-Aldrich). Arabidopsis calmodulin was expressed in High Five TM cells by infecting them with virus expressing Arabidopsis calmodulin and purified using the method of Awata et al.38 (link). Gallus gallus skeletal muscle actin was prepared using method of Spudich and Watt39 (link).
9
Protein-Protein Interaction Profiling
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Co-immunoprecipitation was performed as described previously.44 (link), 45 (link) In brief, pCAG/FLAG-CRADD WT and CRADD-TLIS variant cDNAs or empty vector were co-transfected with pcDNA3.1/V5-PIDD1-DD cDNA in Neuro2a and HEK293T cells for 40–48 hr, after which whole-cell lysates were clarified by centrifugation at 14,000 rpm for 15 min and incubated with anti-FLAG M2 affinity resin (Sigma) for 2 hr with end-over-end mixing at 4°C. Resin beads were pelleted and washed 3× in Tris-buffered saline (pH 7.2) with protease inhibitor cocktail (Roche), phosphatase inhibitor (PhosSTOP, Roche), 1 mM PMSF, and 0.5%–1% Tween-20, before elution with 150 ng/μL 3x-FLAG peptide (Sigma). Input lysates and eluates were denatured in sample buffer at 100°C for 10 min and then immunoblotted with anti-FLAG (Sigma, 1:1,000) and anti-V5 (ThermoFisher, 1:5,000) antibodies.
10
Affinity purification of RACK1 complexes
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