Full-length LAF-1, Whi3, and GAR-1ΔN fragments were tagged with 6×-His and expressed in E. coli using standard procedures. Whi3 purification and BNI1 mRNA transcription were performed following Zhang et al32 (link). Briefly, Whi3 was purified on Ni-NTA (Qiagen) resin and stored in Whi3 elution buffer at −80°C. BNI1 DNA template was obtained using a T7 promoter cloned to the 5’ end of BNI1. DNA template was extracted and transcribed using standard procedures. GAR-1ΔN was purified on Ni-NTA agarose resin (Qiagen) and stored in Ni-elution buffer at −80°C. LAF-1 purification was performed following Elbaum-Garfinkle et al6 (link). LAF-1 was purified on Ni-NTA agarose resin (Qiagen) followed by a HiTrap Heparin column (GE) and stored in Heparin elution buffer. For LAF-1 and GAR-1ΔN, glycerol was added to 10% (vol/vol) before flash freezing in liquid nitrogen and storing at −80°C. See Supplementary Methods for more details.
Ni nta agarose resin
Manufactured by Qiagen
Sourced in Germany, United States, Denmark, Netherlands
Ni-NTA agarose resin is a chromatography material used for the purification of recombinant proteins. It consists of nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. The Ni-NTA functional group binds to histidine-tagged proteins, allowing their capture and purification.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Emulsiflex c5, by Avestin (4 mentions)
Amylose resin, by New England Biolabs (3 mentions)
Superdex 200 gel filtration column, by Cytiva (3 mentions)
Source 15q resin, by Cytiva (3 mentions)
Superdex 200, by GE Healthcare (3 mentions)
Anti gfp antibody, by Rockland Immunochemicals (3 mentions)
Superdex 75 increase 10 300 gl column, by GE Healthcare (3 mentions)
Imidazole, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
10 dg desalting column, by Bio-Rad (2 mentions)
Dnase, by Merck Group (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Pcr2.1 topo vector, by Thermo Fisher Scientific (2 mentions)
Lr clonase 2 enzyme mix, by Thermo Fisher Scientific (2 mentions)
Disposable empty gravity column, by Qiagen (2 mentions)
T4 gt7 dna 165 644 bp, by Nippon Gene (2 mentions)
N trimethoxymethyl silyl propyl n n n trimethyl ammonium chloride, by Gelest (2 mentions)
Biotin peg succinimidyl carbonate, by Laysan Bio (2 mentions)
199 protocols using ni nta agarose resin
1
Purification of LAF-1, Whi3, and GAR-1ΔN Proteins
2
Recombinant Protein Production and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIM32-HIS was expressed from pET28b vector (Clontech Laboratories, Mountain View, CA) and PR8-PB1-GST was expressed from pGEX-5X-3 vector (GE Healthcare Life Sciences, Pittsburgh, PA) in Escherichia coli BL21(DE3) pLysS (Life Technologies) induced with 0.2 mM isopropyl-1-thio-β-D-galactopyranoside and 200 μM ZnSO4 for 16 hr at 18°C as detailed elsewhere [38 (link)]. GST-tagged proteins were purified with glutathione Sepharose 4B beads according to the manufacturer’s protocol (GE Healthcare Life Sciences). HIS-tag proteins were purified with Ni-NTA agarose resins according to the manufacturer’s protocol (Qiagen).
3
Recombinant Lysyl Oxidase Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primer sequences pET21a-LOX F: 5'-CTAGGGGCTAGCGACGACCCTTACAACCCCTA-3' NheI R: 5'-TTACCCAAGCTTATACGGTGAAATTGTGCAGCCTG-3' HindIII
The underlined bold letters indicate the unique restriction sites used for subcloning. LOX, lysyl oxidase; LOX-v2, lysyl oxidase-variant 2.
(amplitude 60). After centrifugation, the inclusion bodies were washed and solubilized in a buffer containing 6 M urea, 10 mM K 2 HPO 4 , pH 8.2, and 3 mM β-mercaptoethanol. The LOX and LOX-v2 recombinant proteins were then purified using the Ni-NTA agarose resins (Qiagen, Hilden, Germany) according to the manufacturer's suggestion. The eluted LOX and LOX-v2 proteins were collected and refolded through stepwise dialysis in a buffer containing 10 mM K 2 HPO 4 , pH 9.6, 200 μM CuCl 2 , and 2% sodium N-lauroylsarcosinate and then in a buffer containing 10 mM K 2 HPO 4 , pH 9.6, and 5 μM CuCl 2 . The protein samples were further dialyzed in 10 mM K HPO 4 , pH 9.6. The dialyzed proteins were lyophilized in the presence of 10 mM trehalose in a freeze dryer (Labconco, Kansas City, MO, USA). All purification and refolding procedures were performed at 4℃. The purity and sizes of the LOX and LOX-v2 proteins were determined by SDS-PAGE.
The underlined bold letters indicate the unique restriction sites used for subcloning. LOX, lysyl oxidase; LOX-v2, lysyl oxidase-variant 2.
(amplitude 60). After centrifugation, the inclusion bodies were washed and solubilized in a buffer containing 6 M urea, 10 mM K 2 HPO 4 , pH 8.2, and 3 mM β-mercaptoethanol. The LOX and LOX-v2 recombinant proteins were then purified using the Ni-NTA agarose resins (Qiagen, Hilden, Germany) according to the manufacturer's suggestion. The eluted LOX and LOX-v2 proteins were collected and refolded through stepwise dialysis in a buffer containing 10 mM K 2 HPO 4 , pH 9.6, 200 μM CuCl 2 , and 2% sodium N-lauroylsarcosinate and then in a buffer containing 10 mM K 2 HPO 4 , pH 9.6, and 5 μM CuCl 2 . The protein samples were further dialyzed in 10 mM K HPO 4 , pH 9.6. The dialyzed proteins were lyophilized in the presence of 10 mM trehalose in a freeze dryer (Labconco, Kansas City, MO, USA). All purification and refolding procedures were performed at 4℃. The purity and sizes of the LOX and LOX-v2 proteins were determined by SDS-PAGE.
4
Purification of Recombinant Proteins by Affinity and Size-Exclusion Chromatography
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The recombinant proteins were purified in two steps: affinity chromatography and SEC. 6His-tagged proteins were affinity-purified using Gravity Flow Chromatography columns (Bio-Rad) loaded with Ni-NTA agarose resin (Qiagen). Strep-tagged proteins were affinity-purified on an ÄKTA Purifier using StrepTrap HP columns (GE Healthcare). Both the 6His- and Strep-tagged proteins were then further purified by SEC on an ÄKTA Purifier using a Superdex 200 16/600 GL column (GE Healthcare), and simultaneously buffer-exchanged into 50 mM HEPES pH 7.5, 150 mM NaCl.
SeMet-labeled PilA and PilC for phasing were purified as above, but the bacteria were grown in chemically defined medium containing SeMet. 15N and/or 13C-labeled PilA for NMR analysis was purified as above, but the bacteria were grown in M9 medium supplemented with 15NH4Cl and/or 13C-labeled glucose.
SeMet-labeled PilA and PilC for phasing were purified as above, but the bacteria were grown in chemically defined medium containing SeMet. 15N and/or 13C-labeled PilA for NMR analysis was purified as above, but the bacteria were grown in M9 medium supplemented with 15NH4Cl and/or 13C-labeled glucose.
5
Purification and Characterization of Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4-Aminoantipyrine and Isopropyl ß-D-1-thiogalactopyranoside (IPTG) from Hi-Media, India; Catechol (extra pure, 99%) from SRL, India and ccMA from Sigma-Aldrich, India were purchased. Restriction endonuclease enzymes and T4 DNA Ligase enzyme were purchased from NEB, United States. pGEM-T Easy Vector Systems (Promega, United States) was used for TA cloning. E. coli DH5α, E. coli BL21 and pET30b(+) vector were procured from the original stock of our lab. Ni-NTA Agarose resin from Qiagen, Germany; Sephacryl S-300 HR resin from Cytiva, United States and NativeMark™ Unstained Protein Standards were obtained from Invitrogen, United States.
6
Protein Extraction and Immunoprecipitation Protocol
7
Purification and Characterization of rh-FAM20C
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant human (rh) FAM20C WT- and D478A-V5/His proteins were generated and purified in the same manner as we previously reported40 (link). Briefly, 293 cells were transfected with the pc3.1-FAM20C WT- or D478A-V5/His vector, and the clones derived from G418-resistant cells were isolated. Conditioned media were collected from each clone (out of 10 clones), and IP-WB analysis was performed with anti-V5 antibody. The clone which synthesized the highest level of rhFAM20C-V5/His protein was further cultured, the conditioned media were collected, and rhFAM20C WT- or D478A-V5/His protein was purified using a Ni-NTA agarose resin (Qiagen). The purified each rhFAM20C protein was pooled, dialyzed against distilled water, lyophilized and dissolved in distilled water. The protein concentration was measured using a DC protein assay kit and it was kept at −20 °C until use. The purity of the rhFAM20C WT- or D478A-V5/His protein was assessed by 4–12% SDS-PAGE stained with CBB and by WB analyses with anti-V5 and anti-FAM20C antibodies. The presence of hFAM20C was also confirmed by Mass Spectrometry analysis (data not shown).
8
Optimized Bacterial Protein Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BL21 (DE3) E.coli cells (EMD Millipore) were transformed with plasmids encoding MBP-LOVSoc or GB1-ORAI1-CT, and grown at 37ºC in LB medium with 100 mg/L of ampicillin. Protein expression was induced by the addition of 500 μM IPTG when OD600 of the culture reached between 0.6 and 0.8. After IPTG induction, MBP-LOVSoc was incubated at 16ºC for additional 6–8 hr, whilst GB1-ORAI1CT incubated at 37ºC for 3–4 hr. Harvested cells were resuspended in 1X Phosphate Buffered Saline (PBS) and sonicated. The cellular debris was clarified by centrifugation. For His6-tagged GB1-ORAI1-CT, the cell lysates were applied to Ni2+-nitrilotriacetic acid (Ni-NTA)-agarose resin (Qiagen). Bound recombinant proteins were eluted in PBS containing 250 mM imidazole and 1 mM TCEP. MBP and MBP-LOVSoc were purified through affinity purification with amylose resin (New England Biolabs) and finally eluted by PBS buffer containing 25 mM maltose and 1 mM TCEP. The proteins were further purified by gel filtration on Superose 6 10/300 GL or Superdex 200 10/300 GL columns (GE Healthcare).
9
Recombinant Protein Expression Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the chemicals and antibiotics were purchased from Sigma (Sigma-Aldrich) and HiMedia laboratories. Ligase, restriction endonucleases, and Taq polymerase were purchased from New England Biolabs (NEB), GST agarose resin was from Gold bioscience, USA, and Ni-NTA agarose resin and QIAquick spin columns were from Qiagen, Germany. Oligonucleotides were custom-synthesized by Imperial Life Sciences (ILS). E. coli strains DH5α (Bethesda Research Laboratories) were used as the host for cloning. E. coli BL 21(DE3) pLysS strain purchased from Novagen and E. coli Top 10 cells from Invitrogen were used in the endogenous assay. Chemical and salts used for protein purification and LB medium used for growing bacterial strains were purchased from HiMedia. Ampicillin, Kanamycin, and Chloramphenicol were used in the concentration of 100, 35, and 25 μg mL−1, respectively.
10
Protein Purification and Refolding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein solution was loaded to a Ni‐NTA agarose resin (Qiagen)‐charged column and washed with 40 volume equivalents of washing buffer. The resin‐bound protein was eluted at 250 mm imidazole elution buffer. The purified protein was dialyzed using a Slide‐A‐Lyzer Dialysis cassettes (Thermo Fisher Scientific, 12 mL, CA; MWCO 10 000 Da) in presence of a refolding buffer (pH 8.2) and dialyzed through a phosphate buffered saline (PBS) at pH of 7.4. Protein was concentrated through a cellulose membrane (MWCO 10 000 Da).
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!