Mouse anti gfp

Cell extracts were made by disruption with glass beads in urea buffer (8 M urea and 1 mM phenylmethylsulphonyl fluoride) followed by 10 min of centrifugation at 21,500× g. Protein concentrations in the supernatants were determined using a Nano-Drop (Thermo-Scientific, Shanghai, China). Then, 100 μg of proteins were subjected to 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Shanghai, China). Mouse anti-FLAG (Transgen, Beijing, China), mouse anti-GFP (Transgen, Beijing, China), mouse anti-HA (Transgen, Beijing, China), or mouse anti-actin (Transgen, Beijing, China) antibodies were used as primary antibodies at 1:5000 dilutions. Goat anti-mouse IgG-HRP (Transgen, Beijing, China) was used as a secondary antibody at 1:5000 dilution. Signals were visualized by Clarity Western ECL Substrate (Bio-Rad, Shanghai, China). Tanon-5200Multi (Tanon, Shanghai, China) was used to obtain Western blot images. Quantification of the signals were performed with ImageJ software [30 ]. Intensities of target protein band were normalized to actin (the intensity of the target protein band was divided by the relative intensity of the corresponding actin band). Then, relative intensities of target protein bands were calculated.

E. coli strains DH5α and BL21 (DE3), and the plasmid pET-22b and pET-28b are products of Novagen (USA). Human cDNA library was supplied by Maikun Teng, University of Science and Technology of China. The genes encoding the EmGFP, mCherry, the codon-optimized Vhb, the mature mSF (V71-S212) with insertion at BamH I and Xho I sites in the expression vectors, and the helper plasmid for coexpressing yeast mature 5-aminolevulinic acid synthase (ALAS) were constructed by our laboratory [24 (link), 25 (link)].
Heparin Sepharose CL-6B, Phenyl Sepharose CL-4B and amylose resin were obtained from GE (Healthcare, USA). Nickel-nitrilotriacetic acid (Ni–NTA) agarose was purchased from Qiagen (Hilden, Germany). Ultra-15 centrifugal filter tubes equipped with the Ultracel-10 membrane were purchased from Merck-Sigma-Aldrich (Kenilworth, NJ, USA). Primers and the gene encoding the CrLOV were synthesized in General Biol Company (Chuzhou, Anhui, China). Monoclonal antibody including mouse anti-GFP, and anti-MBP, and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG antibody, reagents for Western blot analysis were bought from Transgen Biotech (Beijing, China). RAC was prepared from microcrystalline cellulose, according to our previous report [24 (link)].

To extract the proteins from the cells, the cells (20 mg) were suspended in 200 μL of 8 M urea sonicated with a probe-type sonicator on ice. The sonication conditions were as follows: power, 45%, and duration, 60 min, with cycles of 1 min on and 1 min off. The protein concentration was measured with a NanoDrop spectrophotometer (Thermo Scientific), and 100 μg of protein was subjected to 10% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Shanghai). Mouse anti-FLAG (Transgen), mouse anti-GFP (Transgen), or anti-actin antibody (Novus) was used as the primary antibody at a 1:5,000 dilution. Goat anti-mouse IgG-horseradish peroxidase (HRP; Transgen) was used as a secondary antibody at a 1:5,000 dilution. Clarity Western ECL substrate (Bio-Rad) was used to display the bands, and Image Quant LAS 4000 (GE Healthcare) was used to acquire the images.