gradient pre cast polyacrylamide tris glycine gels  (Bio-Rad)

Journal: Parasites & Vectors

Article Title: Interaction of hookworm 14-3-3 with the forkhead transcription factor DAF-16 requires intact Akt phosphorylation sites

doi: 10.1186/1756-3305-2-21

Figure Lengend Snippet: Expression of Ac -FTT-2 and detection with anti-human 14-3-3β antibody . A . Western blot of A. caninum L3 worm lysates. Soluble protein (5 μg) from 65,000 activated and non-activated L3 lysate was separated by 4–20% gradient SDS-PAGE, followed by Western blotting with anti-human 14-3-3β antibody. Lane 1, non-activated L3 extract; lane 2, activated L3 extract. B . Expression of A. caninum V5-tagged recombinant FTT-2 in human embryonic kidney 293 cells. A cDNA encoding full length Ac-ftt-2 was cloned into vector pcDNA3.1/V5-His and transfected into HEK293 cells for expression. After 48 h, 16 μg of total protein was separated by SDS-PAGE, and proteins detected by Western blotting with anti-human 14-3-3β antibody (left panel) or anti-V5 antibody (right panel). Lanes 1 and 3, Ac -FTT-2 transfected; lanes 2 and 4, mock transfected (empty vector). C . Immunoprecipitation of A. caninum V5-tagged recombinant FTT-2 expressed in human embryonic kidney 293 cells. Lysates were prepared 48 h after transfection with pcDNA3.1/V5-His/ Ac-ftt-2 . Mock lysates were made from cells transfected with the empty vector alone. Recombinant Ac -FTT-2 was precipitated by the addition of anti-V5 antibody. Bound sample refers to precipitated beads, and unbound refers to supernatant removed from precipitated beads. Lane 1, Ac-ftt-2 whole lysate only (input); lane 2, Ac-ftt-2 lysate unbound sample; Lane 3, Ac-ftt-2 lysate bound sample; Lane 4, mock lysate unbound sample; Lane 5, mock lysate bound sample.

Article Snippet: Samples were separated at 140 V, 40 mA for 1.5 h in a 4–20% gradient pre-cast Novex Tris-glycine SDS polyacrylamide gel (Invitrogen), and transferred to PVDF membrane for 7.5 min in an iBlot apparatus (Invitrogen).

Techniques: Expressing, Western Blot, SDS Page, Recombinant, Clone Assay, Plasmid Preparation, Transfection, Immunoprecipitation

Journal: Parasites & Vectors

Article Title: Interaction of hookworm 14-3-3 with the forkhead transcription factor DAF-16 requires intact Akt phosphorylation sites

doi: 10.1186/1756-3305-2-21

Figure Lengend Snippet: Effect of phosphorylation site mutation on the interaction of Ac -FTT-2 with Ac -DAF-16 . HEK293 cells were co-transfected with 2 μg of pcDNA3.1V5/ Ac-ftt-2 and 2 μg of wild-type or mutant pCMV4FLAG/ Ac-daf-16 plasmids as above. Mock cells received 4 μg of empty pcDNA3.1/V5-His vector. Cell lysates were prepared 48 h after transfection, and then incubated with anti-FLAG (M2) agarose resin. Immunoprecipations were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane. Top panels , Western blot with anti-human 14-3-3β antibody; Bottom panels , the blot was stripped and re-probed with Ac -DAF-16 antiserum. See text for description of the Ac -DAF-16 mutants.

Article Snippet: Samples were separated at 140 V, 40 mA for 1.5 h in a 4–20% gradient pre-cast Novex Tris-glycine SDS polyacrylamide gel (Invitrogen), and transferred to PVDF membrane for 7.5 min in an iBlot apparatus (Invitrogen).

Techniques: Phospho-proteomics, Mutagenesis, Transfection, Plasmid Preparation, Incubation, SDS Page, Membrane, Western Blot

Journal: Parasites & Vectors

Article Title: Interaction of hookworm 14-3-3 with the forkhead transcription factor DAF-16 requires intact Akt phosphorylation sites

doi: 10.1186/1756-3305-2-21

Figure Lengend Snippet: Absence of Ac -FTT-2 in excretory/secretory products of adult and L3 Ancylostoma caninum . ESP from 6000 non-activated or activated L3, and 10 μg of adult ESP were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane for Western blotting. A . Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, adult ESP; lane 4, lysate (20 μg) of HEK293 expressing Ac -FTT-2. The blot was probed with anti-14-3-3β antibody. B . Control gels. Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, recombinant ASP-1 (80 ng); lane 4, adult ESP; recombinant TIMP-1 (130 ng). Blots were probed with anti-ASP-1 antiserum (left) or anti-TMP-1 antiserum (right).

Article Snippet: Samples were separated at 140 V, 40 mA for 1.5 h in a 4–20% gradient pre-cast Novex Tris-glycine SDS polyacrylamide gel (Invitrogen), and transferred to PVDF membrane for 7.5 min in an iBlot apparatus (Invitrogen).

Techniques: SDS Page, Membrane, Western Blot, Expressing, Control, Recombinant