sypro® ruby protein gel stain  (Lonza)

Journal: bioRxiv

Article Title: Sequence grammar and dynamics of subcellular translation revealed by APEX-Ribo-Seq

doi: 10.1101/2025.05.26.656194

Figure Lengend Snippet: (A) Schematics of constructs for APEX2 fusion protein expression. (B) Immunofluorescence analysis of the indicated proteins (detected by EGFP) and desthiobiotinylated proteins/RNAs (detected with Alexa Fluor 555-conjugated streptavidin). DAPI (4’,6-diamidino-2-phenylindole) was used to stain the DNA. The scale bar represents 10 μm. (C) Western blotting and RNA dot blotting for proteins and RNAs under the indicated conditions. Desthiobiotinylated proteins and RNAs were detected using infrared dye-conjugated streptavidin. As a loading control, RPS17 protein was subjected to Western blotting, and total RNA was stained with methylene blue. (D) Polysome profiling under the indicated conditions. (E) Western blotting and RNA dot blotting for proteins and RNAs along the fractions of polysome profiling (see D for the corresponding fraction numbers). Desthiobiotinylated proteins and RNAs were detected by infrared dye-conjugated streptavidin. Total RNA was stained with methylene blue. (F) Scatter plot of the ribosome footprints from each mRNA in standard Ribo-Seq. The results in the presence and absence of H 2 O 2 were compared. (G) SYPRO Ruby staining of proteins purified after streptavidin pulldown and elution under the indicated conditions. (H) Fractions of usable reads (reads after deduplication and removal of noncoding RNA-mapped reads) in standard Ribo-Seq and cytosol APEX-Ribo-Seq under the indicated conditions. (I) Distribution of ribosome footprint length under the indicated conditions. (J) Metagene plots of ribosome footprints (the 5′-end positions) around the start codon under the indicated conditions. The data for the 29-nt footprints are shown. (K) Fraction of the frame position for the 5′ end of the footprint (29 nt) under the indicated conditions. (L) Scatter plot of the ribosome footprints from each mRNA in cytosol APEX-Ribo-Seq replicates. (M) Immunofluorescence analysis of the indicated proteins (detected by V5) and desthiobiotinylated proteins/RNAs (detected with Alexa Fluor 555-conjugated streptavidin). TOM20 was detected as a mitochondrial marker. The scale bar represents 10 μm. (N) Fraction of the frame position for the 5′ end of the mitoribosome footprint (32 nt) in matrix APEX-Ribo-Seq. TPM, transcripts per million; r , Pearson’s correlation coefficient; RPM, reads per million reads. For H, K, and N, the means (bars), s.d.s (errors), and individual replicates (n = 2, points) are shown.

Article Snippet: The gels were stained with SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific, S12001) and imaged using a Pharos FX imaging system (Bio-Rad).

Techniques: Construct, Expressing, Immunofluorescence, Staining, Western Blot, Control, Purification, Marker

Journal: International Journal of Molecular Sciences

Article Title: Proteomic Analysis and Expression of Selected Genes During the Early Somatic Embryogenesis of Jatropha curcas L.

doi: 10.3390/ijms26136384

Figure Lengend Snippet: Representative 2D IEF-SDS-PAGE protein profiles of J. curcas somatic embryos. The analysis was carried out using ImageMaster TM 2D Platinum software version 7. Molecular masses (kDa) are indicated on the left, and isoelectric points (pI 4–7) are provided above the gel image. ( a ) Protein profile of pre-globular somatic embryos with an average of 654 ± 41 detected spots, and ( b ) protein profile of globular-stage somatic embryos with 552 ± 30 detected spots as indicated by a red cross. The blue and red circles on the gel indicate the protein spots identified through mass spectrometry; the data obtained are presented in . Red circles correspond to unique protein spots detected exclusively at the globular stage, while blue circles indicate protein spots that were at least 1.5-fold more abundant in the embryogenic stage than in the other stages. Numbers correspond to spots identified with ImageMaster TM 2D Platinum version 7. First-dimensional isoelectric focusing was performed using 11 cm IPG strips loaded with 200 µg of total protein. Second-dimension separation was carried out using 12.5% SDS-PAGE polyacrylamide gels (CRITERION Cell, Bio-Rad Laboratories, Hercules, CA, USA). Gels were stained with Sypro Ruby (Bio-Rad Laboratories, Hercules, CA, USA).

Article Snippet: Following electrophoresis, the gels were fixed in 10% ( v / v ) ethanol and 7% ( v / v ) acetic acid for 30 min and subsequently stained with Sypro Ruby protein stain (Bio-Rad Laboratories, Hercules, CA, USA) for 20 h. Destaining was performed by incubating the gels in the same fixing solution for 1 h with gentle agitation.

Techniques: SDS Page, Software, Mass Spectrometry, Staining