profinity exacttm purification resin  (Bio-Rad)

Journal: Scientific Reports

Article Title: Activation of hepatic stellate cells by the ubiquitin C-terminal hydrolase 1 protein secreted from hepatitis C virus-infected hepatocytes

doi: 10.1038/s41598-017-04259-7

Figure Lengend Snippet: UCHL1 induced HSCs activation. ( A ) HSC-T6 cells (1 × 10 6 cells) were co-cultured with Con1 or Huh7 cells (1 × 10 5 cells) in the presence or absence of anti-UCHL1 antibody or mouse IgG control antibody for 24 h. The HSC-T6 cell lysate was collected and the expression of α-SMA was determined by Western blot analysis. HSC-T6 cells treated with DMEM and TGF-β1 were used as the negative and positive control of HSC activation, respectively. The expression of β-tubulin was used as the control for equal protein loading. The number below the gel images represents the relatively α-SMA expression levels for the indicated experimental condition after normalization with the expression of β-tubulin. ( B ) Recombinant UCHL1 protein fused to the Profinity eXact tag was expressed in E . coli BL21 after IPTG induction. Samples from the indicated purification steps were fractionated on a 12% SDS-PAGE and stained with Coomassie Brilliant Blue (upper panel). The protein with an expected molecular weight of UCHL1 (21 kDa) was noted in the eluted fraction after protease cleavage of the Profinity eXact tag and was indicated as *. The recombinant UCHL1 protein in the indicated steps of protein purification was further confirmed by Western blotting using the anti-UCHL1 antibody (lower panel). The cell lysate of Con1 was used as a positive control. ( C ) HSC-T6 cells were culture in DMEM w/o FBS for 24 h and then treated with the indicated concentrations of purified rUCHL1 or BSA for an additional 24 h. The cell lysates were collected and the expression of α-SMA was determined by Western blot analysis. The expression of β-tubulin was used as the control for equal protein loading. The number below the gel images represents the relatively α-SMA expression levels for the indicated experimental condition after normalization with the expression of β-tubulin. HSCs treated with DMEM and TGFβ1 (10 ng/ml) were used as negative and positive controls for HSC stimulation, respectively. The full Western blot and the corresponding positions of the molecular weight protein markers are presented in Supplementary Fig. .

Article Snippet: The supernatant rUCHL1 protein was purified by using the Profinity eXactTM Protein Purification Systems (Bio-Rad, Richmond, CA) and was concentrated using an Amicon Ultra-4 ® 10 K (Millipore, Bedford, MA) for further analysis.

Techniques: Activation Assay, Cell Culture, Control, Expressing, Western Blot, Positive Control, Recombinant, Purification, SDS Page, Staining, Molecular Weight, Protein Purification

Journal: Methods in Molecular Biology

Article Title: Heterologous Gene Expression in E.coli

doi: 10.1007/978-1-4939-6887-9

Figure Lengend Snippet: Fig. 1 Schematic representation of the Profinity eXact™ vectors. The Bio-Rad vectors pPAL7 and pPAL8 harbor only an N-terminal Profinity eXact™ tag (green) followed by a multiple cloning site (MCS, orange). The other vectors were constructed in-house and are based on the pET vector backbone (Novagen) to contain 6× His-tag (marked in red) with or without an additional expression enhancing tag (blue). The components of the expres- sion cassette are not drawn to scale. The MCS and the antibiotic resistance in the Profinity-pET based vectors differ from the ones in pPAL7 and pPAL8 (see Note 4)

Article Snippet: Mix the Profinity eXactTM affinity resin and transfer 2 mL of resin suspension (equivalent to 1 mL of settled beads) to the Bio-Rad Econo-Pac column.

Techniques: Cloning, Construct, Plasmid Preparation, Expressing

Journal: Methods in Molecular Biology

Article Title: Heterologous Gene Expression in E.coli

doi: 10.1007/978-1-4939-6887-9

Figure Lengend Snippet: Fig. 2 Comparison of protein expression using different solubility tags. The Water Soluble Chlorophyll Protein gene (WSCP, amino acids 12–190, NCBI XP_013613804.1) was cloned into each of the vectors listed in Fig. 1 (see Note 5). Expression of proteins was performed at 37 °C for 3 h. Cell pellets were pro- cessed in parallel, as described in the text. Analysis was performed using Bolt 4–12% Bis-Tris plus gel (Invitrogen). S- Soluble fraction, following cell lysis. E- Elution fraction following cleavage from the Profinity eXact™ tag. Arrow indi- cates position of the WSCP following cleavage. Asterisk indicates position of the full-length fusion protein in each of the soluble fraction

Article Snippet: Mix the Profinity eXactTM affinity resin and transfer 2 mL of resin suspension (equivalent to 1 mL of settled beads) to the Bio-Rad Econo-Pac column.

Techniques: Comparison, Expressing, Solubility, Clone Assay, Lysis