Cells were washed twice with 1x PBS, scraped up on ice, and transferred to 1.7 ml tubes followed by pelleting by centrifugation whereupon supernatant was removed. Cells were lysed for 30 minutes in 500 µL of co-immunoprecipitation buffer (200 mM KCL, 25 mM Hepes, 1% NP-40, 20 mM NaF, 1 mM Na-orthovanadate, 0.2 mM EGTA, 1x protease arrest [G Biosciences], 1x phosphatase inhibitor cocktail II [Alfa Aesar], 20 µM nicotinamide, pH 7.5) and sonicated. Lysates were then subjected to centrifugation and whole cell lysate samples were generated using the 50 µL of the total lysate, to which 4x SDS page lysis buffer was added, followed by vortexing and boiling for 5 minutes. For immunoprecipitation of FLAG tagged proteins, the remaining 450 µL of lysate was incubated with 20 µL of anti-FLAG G1 affinity resin (GenScript) overnight on a tube rotator at 4 °C. After incubation, washing was performed to remove non-specific binding contaminates. Briefly, samples were centrifuged, supernatant removed, washed with 500 µL of co-immunoprecipitation buffer, and allowed to rotate at 4 °C on a tube rotator for 5 minutes. These steps were repeated three times, before a final centrifugation and removal of supernatant, after which 50 µL of 1x SDS page lysis buffer was added to the pellets before vortexing and boiling for 5 minutes.
Anti flag g1 affinity resin
Manufactured by GenScript
Sourced in China
Anti-Flag G1 affinity resin is a chromatography resin designed for the purification of Flag-tagged proteins. It is composed of an agarose base matrix and immobilized anti-Flag G1 antibody fragments, which specifically bind to the Flag epitope tag.
Automatically generated - may contain errors
Lab products found in correlation
Flag peptide, by GenScript (3 mentions)
Protease arrest, by G Biosciences (1 mentions)
Resource q anion exchange column, by GE Healthcare (1 mentions)
Ni nta column, by Qiagen (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Benzonase, by Merck Group (1 mentions)
Centricon, by Merck Group (1 mentions)
Source 15q 10 100, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300, by GE Healthcare (1 mentions)
Superose 6 increase 10 300 gl column, by GE Healthcare (1 mentions)
Hitrap heparin hp column, by GE Healthcare (1 mentions)
Protease inhibitor cocktail c0001, by Targetmol (1 mentions)
Anti ha magnetic beads, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Protein g sepharose, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
9 protocols using anti flag g1 affinity resin
1
Immunoprecipitation of FLAG-Tagged Proteins
2
Purification of Tubulin Variants
3
Immunoprecipitation of Flag-tagged NS1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For candidate proteins that interacted with the NS1 protein, the pCI-optNS1Flag or pCI vector plasmid was transfected to HEK293 cells cultured in a 145-mm dish, and at 2 days after transfection, the cells were washed twice with cold PBS and lysed with 1 mL lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, and protease inhibitors) by mixing with discontinuous agitations for 30 min on ice. The cell lysates were treated with or without 250 U of Benzonase (number E8263; Sigma), followed by centrifugation at 12,000 rpm for 10 min at 4°C. The supernatant was then collected, of which 80 μL was boiled in loading buffer as an input control, and the remaining supernatant was incubated with 100 μl of prewashed anti-Flag G1 affinity resin (number L00432; GenScript, Piscataway, NJ) followed by rotation at 4°C overnight. Finally, the beads were washed 3 times with washing buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, and 1 mM EDTA) for 3 min before mixing with 2× Laemmli loading buffer for Western blotting.
4
Affinity Purification of SAMP1-MoaE Conjugate
Check if the same lab product or an alternative is used in the 5 most similar protocols
H. volcanii cells were harvested by centrifugation (8,000 g, 10 min, 25°C) and washed twice with TBS buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4). Cell pellets were stored at -20°C until further use. The harvested cells were lysed by French press homogenization (12,000 lb/in2) in lysis buffer composed of TBS supplemented with 0.1 mM CaCl2, 2 mM MgCl2, DNase I and protease inhibitor cocktail. Cell debris was removed by centrifugation (20,000 g, 40 min, 4°C) and filtration (0.22 μm) and the clarified cell lysate was then applied to an anti-Flag column pre-equilibrated with TBS. The column was 1 cm in diameter and was filled with 0.2–0.4 ml anti-Flag G1 affinity resin (Genscript). The supernatant was incubated with resin at room temperature for 1 h. The bound proteins were washed in 15 mL TBS prior to elution in 100 μl 2X reducing SDS-PAGE loading buffer. The purified Flag-tagged proteins were separated on 12% reducing SDS-PAGE gel and stained with Coomassie G250 dye. The protein band representing SAMP1(SAMP1UAG24)-MoaE conjugate at 50 kDa was cut for further analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS).
+ Expand
H. volcanii cells were harvested by centrifugation (8,000 g, 10 min, 25°C) and washed twice with TBS buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4). Cell pellets were stored at -20°C until further use. The harvested cells were lysed by French press homogenization (12,000 lb/in2) in lysis buffer composed of TBS supplemented with 0.1 mM CaCl2, 2 mM MgCl2, DNase I and protease inhibitor cocktail. Cell debris was removed by centrifugation (20,000 g, 40 min, 4°C) and filtration (0.22 μm) and the clarified cell lysate was then applied to an anti-Flag column pre-equilibrated with TBS. The column was 1 cm in diameter and was filled with 0.2–0.4 ml anti-Flag G1 affinity resin (Genscript). The supernatant was incubated with resin at room temperature for 1 h. The bound proteins were washed in 15 mL TBS prior to elution in 100 μl 2X reducing SDS-PAGE loading buffer. The purified Flag-tagged proteins were separated on 12% reducing SDS-PAGE gel and stained with Coomassie G250 dye. The protein band representing SAMP1(SAMP1UAG24)-MoaE conjugate at 50 kDa was cut for further analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS).
5
Purification of UVR8 Protein Mutant
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The stored cell suspension was thawed at room temperature and lysed using a JN-02 homogenizer (JNBIO, China). The lysed cells were ultracentrifuged at 20 000 × g at 4°C for 1 h to remove the cell pellets. The supernatant was incubated with anti-Flag G1 affinity resin (GenScript) at 4°C for 2 h. The bound proteins were washed with 5 column volumes (CVs) of buffer A and then eluted with buffer A containing 300 μg ml−1 Flag peptide (Genscript). The eluted protein was further purified by anion-exchange chromatography (Source 15Q 10/100, GE Healthcare). The protein was concentrated with a 10-kDa cutoff Centricon (Millipore) and then subjected to size-exclusion chromatography (Superdex-200 Increase 10/300, GE Healthcare) in buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 5 mM dithiothreitol (DTT). The homogeneous peak fractions of RUP2-UVR8W285A were pooled and immediately used for crystallization.
For in vitro biochemical assays, the wild-type and mutant UVR8 genes were subcloned into a modified pET15 vector with an N-terminal 6×His tag followed by a drICE protease cleavage site. The fusion proteins were expressed in Escherichia coli cell strain BL21 (DE3) and purified as described previously (Ma et al., 2020b (link)). The proteins were digested by drICE protease before size-exclusion chromatography.
For in vitro biochemical assays, the wild-type and mutant UVR8 genes were subcloned into a modified pET15 vector with an N-terminal 6×His tag followed by a drICE protease cleavage site. The fusion proteins were expressed in Escherichia coli cell strain BL21 (DE3) and purified as described previously (Ma et al., 2020b (link)). The proteins were digested by drICE protease before size-exclusion chromatography.
6
Purification of COP1-SPA4 Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The COP1-SPA41–464 complex was extracted from stored cell pellets. The cell suspension was thawed and lysed using a JN-02 homogenizer (JNBIO, China). The lysed cells were ultracentrifuged at 14,000 rpm at 4°C for 1 hour to remove the insoluble component. The supernatant was incubated with an anti-Flag G1 affinity resin (GenScript) at 4°C for 2 hours. The resin was washed with 20-bed volumes of buffer A and eluted with buffer A with 3×Flag peptide (300 μg ml−1) (GenScript). The eluted protein was further purified by a HiTrap Heparin HP column (GE Healthcare). The protein was concentrated with a 50-kDa-cutoff Centricon (Millipore) and further subjected to Superose 6 Increase 10/300 GL column (GE Healthcare) using the size exclusion chromatography A (SEC-A) buffer containing 25 mM tris-HCl (pH 8.0), 150 mM NaCl, and 5 mM dithiothreitol (DTT). The peak fractions were pooled and immediately used for the reconstitution of COP1-SPA41–464-UVR8 or COP1-SPA41–464-HY5 complexes, in which the UVR8 and HY5 were expressed in E. coli cell strain BL21 (DE3) and purified as previously described (stored in the SEC buffer) (49 (link)).
7
Co-Immunoprecipitation of MFN2 and EGFR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in ice‐cold lysis buffer (50 mmol/L Tris‐HCl [pH 7.5], 150 mmol/L NaCl, 1% Triton X‐100, 1 mmol/L EDTA) with 1 × protease inhibitor cocktail (C0001, TargetMol, Boston, MA, USA) for 30 min. Heterologously expressed MFN2‐Flag and EGFR‐HA were subjected to anti‐Flag G1 affinity resin (L00432, GenScript, Nanjing, Jiangsu, China) or anti‐HA Magnetic Beads (#88836, Thermo‐Fisher Scientific, Waltham, MA, USA). The precipitated proteins were eluted by 400 μg/mL Flag peptide (RP10586, GenScript) or 1 mg/mL HA peptide (TP1276, TargetMol). For co‐IP of endogenous MFN2, cell lysates were incubated with anti‐MFN2 antibody (1‒2 μg) overnight at 4°C. Subsequently, the pre‐cleared protein A/G magnetic beads (#26162, Thermo‐Fisher Scientific) were added to capture the protein complexes. In both cases, the beads were extensively washed at least five times with lysis buffer to remove non‐specifically associated proteins. Eluted proteins were boiled for 10 min in 1 × sodium dodecyl sulphate (SDS) loading buffer and then analyzed by Western blotting. Protein samples were loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a 0.45 μm polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA). After blocking, proteins were immunoblotted with the indicated antibodies.
8
Immunoprecipitation of Flag-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells were transfected with pCI-NP1Flag and vector control. At 2 days post-transfection, the cells were washed twice with cold PBS and lysed with lysis buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, and Protease Inhibitor Cocktail (#S8830, SIGMAFAST, MilliporeSigma)] by constant agitation for 30 min at 4°C. Then, the cell lysates were treated with 250 units of Benzonase nuclease (#E1014-5KU, MilliporeSigma) and centrifugated at 12,000 g for 15 min at 4°C. The supernatant was collected and incubated with 40 μl of prewashed anti-Flag G1 affinity resin (GenScript, Piscataway, NJ) with rotation for ~4 h at 4°C. The beads were then pelleted down by centrifugation at 2,000 g for 3 min, followed by washing with Wash buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, and 1 mM EDTA) three times. The washed beads were mixed with 2 x Laemmli loading buffer and boiled at 95°C for 5 min to elute bound proteins for Western blotting.
9
Immunoprecipitation of KRIT1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human Umbilical Vein Endothelial Cells (HUVEC) were collected in cold lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1%NP40, and 10% glycerol) plus protease and phosphatase inhibitor cocktails (Roche). A total of 2 µg of monoclonal anti-KRIT1 (15B2) antibody was added to 350 µg of clarified lysates and incubated at 4°C overnight while rotating. Protein G-Sepharose (Invitrogen) was added to the reaction mixture and further incubated for 4 hours at 4°C. After three washes with cold lysis buffer, beads were mixed with sample buffer and proteins were separated by SDS-PAGE. Bound KRIT1 was detected by using polyclonal anti-KRIT1 (6832) antibody.
HEK293T or U2OS cells, transfected and infected as indicated, were collected in cold lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1%NP40, and 10% glycerol) plus protease and phosphatase inhibitor cocktails (Roche) and subsequently, clarified lysate was incubated with anti-FLAG G1 Affinity Resin (Genscript, Piscataway, NJ; L00432-1) and incubated at 4°C for 3 hours while rotating. After three washes with cold lysis buffer, beads were mixed with sample buffer and proteins were separated by SDS-PAGE.
HEK293T or U2OS cells, transfected and infected as indicated, were collected in cold lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1%NP40, and 10% glycerol) plus protease and phosphatase inhibitor cocktails (Roche) and subsequently, clarified lysate was incubated with anti-FLAG G1 Affinity Resin (Genscript, Piscataway, NJ; L00432-1) and incubated at 4°C for 3 hours while rotating. After three washes with cold lysis buffer, beads were mixed with sample buffer and proteins were separated by SDS-PAGE.
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!