The epitope-tagging strategy to isolate iASPP or CEP55-containing protein complexes from human cells was performed essentially as previously described with some modifications44. In brief, to obtain a FLAG-HA-iASPP (or CEP55) expressing cell line, HeLa cells were transfected with pCIN4-FLAG-HA-iASPP (or CEP55) constructs and selected for 2 weeks in 1 mg/ml G418. The tagged iASPP protein levels were detected by WB analyses. The stable cell lines were chosen to expand for protein complex purification. For purification, the cells were lysed in BC100 buffer (20 mM Tris-Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100 and fresh protease inhibitor on ice for 2 h. The homogenate was centrifuged for 30 min at 12,000 rpm at 4 °C. Cleared lysates were filtered through 0.45 μM spin filters (Millipore) and immunoprecipitated by anti-FLAG antibody-conjugated M2 agarose (Sigma). The bound polypeptides eluted with the FLAG peptide (Sigma) were further affinity purified by anti-HA antibody-conjugated agarose (Sigma). The final elutes from the HA-beads with HA peptides were resolved by SDS-PAGE on a 4–20% gradient gel (Bio-Rad) for Coomassie Blue staining. Gel bands were cut out from the gel and subjected to mass-spectrometric sequencing.
Anti flag antibody conjugated m2 agarose
Manufactured by Merck Group
Sourced in United States
The Anti-FLAG antibody-conjugated M2 agarose is a laboratory product used for the purification and detection of proteins tagged with the FLAG peptide sequence. It consists of an agarose resin with immobilized anti-FLAG monoclonal antibodies, which bind to the FLAG tag on the target protein, allowing for its isolation and enrichment from complex samples.
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Lab products found in correlation
Flag peptide, by Merck Group (3 mentions)
Anti ha antibody conjugated agarose, by Roche (3 mentions)
Complete protease inhibitor cocktail mix, by Roche (3 mentions)
0.45 μm spin filters, by Merck Group (2 mentions)
Anti ha antibody conjugated agarose, by Merck Group (2 mentions)
Gradient gel, by Bio-Rad (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Na931v, by GE Healthcare (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Chaps, by Carl Roth (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ha peptide, by Roche (1 mentions)
Mascot search engine, by Matrix Science (1 mentions)
150 mm dishes, by Corning (1 mentions)
10 protocols using anti flag antibody conjugated m2 agarose
1
Affinity Purification of Epitope-Tagged Protein Complexes
2
Isolation of GLI1-containing Protein Complexes
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The epitope-tagging strategy to isolate GLI1-containing protein complexes from human cells was performed essentially as described previously55 (link). HEK293 cells infected with HA-FLAG-Gli1 recombinant retrovirus were grown in 293SFM medium and harvested at near confluency (~1 × 109 cells). The cells were separated into cytoplasmic and nuclear fractions. Cytoplasmic fraction was dialysed for 5 h in a buffer consisting of 20 mM Hepes–KOH (pH 7.9), 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT. The lysate was centrifuged at 15,000×g for 15 min at 4 °C. The supernatants were immunoprecipitated with anti-FLAG antibody-conjugated M2 agarose (400 μl; Sigma-Aldrich). Bound proteins eluted 0.4 mg ml−1 FLAG peptide (Sigma-Aldrich) were further affinity purified using 20 μl anti-HA antibody-conjugated agarose (Roche Diagnostics). The final elutes from HA beads eluted with 2.5 mg ml−1 HA peptide (Roche Diagnostics) were separated by SDS–PAGE on a 5–20% gradient gel for silver staining analysis. Specific bands were excised from the gel and subjected to peptide sequencing by mass spectrometry using a 4700 proteomics analyser (AB SciEx). Data were analysed by the Mascot search engine (Matrix Science).
3
Identification of ID2 Protein Interactome
4
Purification of Borealin Protein Complex
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HeLa cells were transfected with pCIN4-Flag-HA-Borealin constructs and selected for 3 weeks in 1.5 mg/mL G418. The tagged Borealin protein levels were detected by western blot. The stable cell lines that express Flag-HA-Borealin were chosen to expand. For protein complex purification, the HeLa/Borealin cells were lysed in BC100 buffer (20 mM Tris-Cl, pH 7.5, 20% glycerol, 100 mM NaCl, and 0.2 mM EDTA) containing 0.1% Triton X-100 and protease inhibitor on ice for 4 h. After centrifugation for 40 min with 12500 rpm at 4°C, the supernatant of the cell lysates was immunoprecipitated by anti-Flag antibody-conjugated M2 agarose (Sigma). After washing three times with the BC100 buffer to remove the unbounded protein, immunoprecipitated by anti-HA antibody-conjugated M2 agarose (Sigma), the bound polypeptides were eluted with peptide and resolved by SDS-PAGE for Coomassie Blue staining. Gel lanes (strips) were subjected to mass spectrometric sequencing.
5
Immunoprecipitation of Salmonella Effectors
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HeLa cells grown in 150-mm dishes (Corning, United Kingdom) were infected with S. Typhimurium for 14 h. Phosphate-buffered saline (PBS)-washed cells were scraped with a rubber policeman, centrifuged for 5 min at 100 × g, and resuspended in 500 µl of lysis buffer (PBS–5% glycerol–0.5% Triton X-100–1 mM phenylmethylsulfonyl fluoride [PMSF]) for 30 min at 4°C. Lysates were centrifuged for 10 min at 16,000 × g to remove debris before being precleaned with 20 µl of protein G agarose for 1 h (Pierce). The precleaned lysate was mixed with 40 µl of anti-HA antibody-conjugated agarose (Pierce) or anti-Flag antibody-conjugated M2 agarose (Sigma) and incubated at 4°C for 2 h to immunoprecipitate HA-tagged SseF or Flag-tagged SseG. The immunoprecipitates were washed 4 times with lysis buffer and then eluted with 50 µl of 2 mg/ml HA peptide or 0.1 mg/ml Flag peptide. Sample proteins were separated by SDS-PAGE and analyzed by immunoblotting with appropriate antibodies.
6
Identification of ID2 Protein Interactome
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Cellular ID2 complexes were purified from the cell line NCI-H1299 stably engineered to express FLAG-HA-ID2. Cellular lysates were prepared in 50 mM Tris-HCl, 250 mM NaCl, 0.2% NP40, 1 mM EDTA, 10% glycerol, protease and phosphatase inhibitors. FLAG-HA-ID2 immunoprecipitates were recovered first with anti-FLAG antibody-conjugated M2 agarose (Sigma) and washed with lysis buffer containing 300 mM NaCl and 0.3% NP40. Bound polypeptides were eluted with FLAG peptide and further affinity purified by anti-HA antibody-conjugated agarose (Roche). The eluates from the HA beads were analyzed directly on long gradient reverse phase LC-MS/MS. A specificity score of proteins interacting with ID2 was computed for each polypeptide by comparing the number of peptides identified from our mass spectrometry analysis to those reported in the CRAPome database that includes a list of potential contaminants from affinity purification-mass spectrometry experiments (www.crapome.org ). The specificity score is computed as [(#peptide*#xcorr)/(AveSC*MaxSC* # of Expt.)], #peptide, identified peptide count; #xcorr, the cross-correlation score for all candidate peptides queried from the database; AveSC, averaged spectral counts from CRAPome; MaxSC, maximal spectral counts from CRAPome; and # of Expt., the total found number of experiments from CRAPome.
7
Purification of ASPP1/ASPP2 Protein Complexes
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The epitope-tagging strategy to isolate ASPP1 (or ASPP2)-containing protein complexes from human cells was performed essentially as previously described with some modifications [45 (link)]. In brief, to obtain a FLAG-HA-ASPP1 (or ASPP2) expressing cell line, HeLa cells were transfected with pCIN4-FLAG-HA-ASPP1 (or ASPP2) constructs and selected for 2 weeks in 1 mg/ml G418. The tagged ASPP1 or ASPP2 protein levels were detected by WB analyses. The stable cell lines were chosen to expand for protein complex purification. For purification, the MOCK, HeLa/ASPP1, or HeLa/ASPP2 cells were lysed in BC100 buffer (20 mM Tris-Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100 and fresh protease inhibitor on ice for 2 hr. The homogenate was centrifuged for 30 min at 12000 rpm at 4°C. Cleared lysates were filtered through 0.45 μM spin filters (Millipore) and immunoprecipitated by anti-FLAG antibody-conjugated M2 agarose (Sigma). The bound polypeptides eluted with the FLAG peptide (Sigma) were further affinity purified by anti-HA antibody-conjugated agarose (Sigma). The final elutes from the HA-beads with HA peptides were resolved by SDS-PAGE on a 4%–20% gradient gel (Bio-Rad) for Coomassie Blue staining. Gel bands were cut out from the gel and subjected to mass-spectrometric sequencing.
8
Co-immunoprecipitation of Protein Complexes
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The coimmunoprecipitation experiments were carried out essentially as previously described.35 (link) Briefly, cells were lysed 24 h after transfection with 600 μl CHAPS lysis buffer consisting of 10 mM CHAPS, 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM NaF, 1 mM DTT, 0.5 mM PMSF and 40 μl/ml 'Complete Mix' protease inhibitor cocktail (Roche, Basel, Switzerland). Extracts were incubated overnight with 40 μl agarose-conjugated anti-Flag antibody (M2, Sigma-Aldrich, St Louis, MO, USA) at 4 °C. Precipitates were washed 6 to 8 times with CHAPS lysis buffer and finally resuspended in SDS-polyacrylamide gel loading buffer. Proteins were resolved via SDS-PAGE and analyzed via western blotting.
9
Immunoprecipitation and Western Blot Analysis
10
Immunoprecipitation and Western Blot Analysis
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HEK293 cells were transfected with the indicated constructs for expression of GFP- and Flag-tagged WT and mutant proteins. 24 hours after transfection cells were lysed with 600 μl CHAPS lysis buffer [10 mM 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS, Roth), 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM NaF, 1 mM Dithiothreitol (DTT, Merck), 0.5 mM Phenylmethanesulfonyl fluoride (PMSF, Merck) and 40 μl/ml “Complete Mix” protease inhibitor cocktail (Roche)]. The extracts were incubated with 40 μl agarose-conjugated anti-Flag antibody (M2, Sigma) at 4°C overnight. Precipitates were washed 6 to 8 times with CHAPS lysis buffer and finally resuspended in SDS-polyacrylamide gel loading buffer. For western blotting the proteins were resolved in SDS-polyacrylamide gels and transferred electrophoretically at room temperature to PVDF membranes (Merck) for 1 h at 50 mA using a Tris-glycine buffer system. The membranes were pre-blocked for 1 h in a solution of 3% milk powder in PBS-T (0.1% Tween 20 in PBS) before adding antibodies. The following antibodies were used: anti-GFP (7.1/13.1, mouse monoclonal IgG, secondary antibody peroxidase conjugated sheep anti-mouse IgG, NA931V, GE healthcare) or anti-Flag (M5, Sigma; secondary antibody, NA931V, GE healthcare).
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