gfp jl 8 clontech 1 1000  (TaKaRa)

Journal: Plant Direct

Article Title: Identification and characterization of the COPII vesicle‐forming GTPase Sar1 in Chlamydomonas

doi: 10.1002/pld3.614

Figure Lengend Snippet: Ectopic expression of the CrSar1DN‐YFP affects protein secretion. (a) Confocal microscopic images of the Sec Sar1 strain co‐expressing CrSar1‐YFP (green) and signal sequence (SS)‐mCherry‐SP 10 (magenta). Chlorophyll fluorescence is shown in cyan. No intracellular mCherry fluorescence signal was observed, suggesting efficient secretion of SS‐mCherry‐SP 10 to the extracellular medium. Scale bar, 5 μm. (b) Confocal microscopic images of the Sec Sar1DN strain co‐expressing CrSar1DN‐YFP (green) and SS‐mCherry‐SP 10 (magenta). Chlorophyll fluorescence is shown in cyan. A strong intracellular mCherry fluorescence signal is observed, suggesting that secretion of SS‐mCherry‐SP 10 is affected by the ectopic expression of CrSar1DN‐YFP. Scale bar, 5 μm. (c) Western blot analysis of protein secretion efficiency in the Sec Sar1DN strain. Secretion efficiency is determined by comparing the relative abundance of the secreted (in the medium fraction, M) and retained (in the cell pellet fraction, P) SS‐mCherry‐SP 10 . Total protein was isolated from both the culture medium and the cell pellet and subjected to separation by sodium dodecyl–sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), followed by immunoblotting using anti‐GFP (for detection of CrSar1‐YFP and CrSar1DN‐YFP) or anti‐RFP (for detection of SS‐mCherry‐SP 10 ) antibodies. SS‐mCherry‐SP 10 was predominately detected in the medium fraction in the Sec Sar1 strain while the mCherry signal was detected in both the medium and cell pellet of the Sec Sar1DN strain. Asterisks indicate the fully glycosylated SS‐mCherry‐SP 10 protein. Bands with lower molecular weights represent non‐glycosylated, partially glycosylated or partially degraded proteins.

Article Snippet: The membranes were then incubated with blocking buffer (5% milk powder in 1× TBS‐T) at room temperature for 1 h. After several washing steps with TBS‐T, the membranes were incubated with the primary antibody at 4°C overnight using the following antibody dilutions: anti‐GFP (JL‐8; Clontech, 1:1000), anti‐RFP (6G6, Chromotek, 1:2000).

Techniques: Expressing, Sequencing, Fluorescence, Western Blot, Isolation, Polyacrylamide Gel Electrophoresis, SDS Page