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Analysis of SP-EVs fractions by (a) transmission electron microscopy (TEM) and (b, c) nanoparticle tracking analysis (NTA, n = 4). (d) Protein concentration (μg/μL) (n = 9) was assessed by Abs280 nm. <t>(e)</t> <t>SDS-PAGE</t> (15%) protein profile of SP-EV preparations and SP (60 μg protein/lane) (n = 3) stained with Coomassie brilliant blue. (f) Western blot analysis (60 μg protein/lane) of EV preparations and complete SP using antibodies against EV-associated proteins CD9, CD63, and HSP70, endoplasmic reticulum marker calnexin (CNX), and a non-vesicular extracellular particle apolipoprotein C-III (ApoC3) (n = 3) (full western blot images in Supplementary Figure 2). SP: seminal plasma; P1–2: pool of fractions in size-exclusion chromatography; CL: cell lysate; Mw: molecular weight marker. Different letters (a, b) above the bars indicate statistically significant differences (p < 0.05) between methods (C, P1, and P2), as determined by a linear mixed-effects model and Tukey-adjusted pairwise comparisons. A significant positive correlation was observed between particle concentration and protein concentration across samples (Spearman’s ρ = 0.747, p = 0.0009). All data are expressed as mean ± SD of four independent experiments (n = 4). Scale bar = 200 nm.
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Analysis of SP-EVs fractions by (a) transmission electron microscopy (TEM) and (b, c) nanoparticle tracking analysis (NTA, n = 4). (d) Protein concentration (μg/μL) (n = 9) was assessed by Abs280 nm. <t>(e)</t> <t>SDS-PAGE</t> (15%) protein profile of SP-EV preparations and SP (60 μg protein/lane) (n = 3) stained with Coomassie brilliant blue. (f) Western blot analysis (60 μg protein/lane) of EV preparations and complete SP using antibodies against EV-associated proteins CD9, CD63, and HSP70, endoplasmic reticulum marker calnexin (CNX), and a non-vesicular extracellular particle apolipoprotein C-III (ApoC3) (n = 3) (full western blot images in Supplementary Figure 2). SP: seminal plasma; P1–2: pool of fractions in size-exclusion chromatography; CL: cell lysate; Mw: molecular weight marker. Different letters (a, b) above the bars indicate statistically significant differences (p < 0.05) between methods (C, P1, and P2), as determined by a linear mixed-effects model and Tukey-adjusted pairwise comparisons. A significant positive correlation was observed between particle concentration and protein concentration across samples (Spearman’s ρ = 0.747, p = 0.0009). All data are expressed as mean ± SD of four independent experiments (n = 4). Scale bar = 200 nm.
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Analysis of SP-EVs fractions by (a) transmission electron microscopy (TEM) and (b, c) nanoparticle tracking analysis (NTA, n = 4). (d) Protein concentration (μg/μL) (n = 9) was assessed by Abs280 nm. <t>(e)</t> <t>SDS-PAGE</t> (15%) protein profile of SP-EV preparations and SP (60 μg protein/lane) (n = 3) stained with Coomassie brilliant blue. (f) Western blot analysis (60 μg protein/lane) of EV preparations and complete SP using antibodies against EV-associated proteins CD9, CD63, and HSP70, endoplasmic reticulum marker calnexin (CNX), and a non-vesicular extracellular particle apolipoprotein C-III (ApoC3) (n = 3) (full western blot images in Supplementary Figure 2). SP: seminal plasma; P1–2: pool of fractions in size-exclusion chromatography; CL: cell lysate; Mw: molecular weight marker. Different letters (a, b) above the bars indicate statistically significant differences (p < 0.05) between methods (C, P1, and P2), as determined by a linear mixed-effects model and Tukey-adjusted pairwise comparisons. A significant positive correlation was observed between particle concentration and protein concentration across samples (Spearman’s ρ = 0.747, p = 0.0009). All data are expressed as mean ± SD of four independent experiments (n = 4). Scale bar = 200 nm.
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Analysis of SP-EVs fractions by (a) transmission electron microscopy (TEM) and (b, c) nanoparticle tracking analysis (NTA, n = 4). (d) Protein concentration (μg/μL) (n = 9) was assessed by Abs280 nm. (e) SDS-PAGE (15%) protein profile of SP-EV preparations and SP (60 μg protein/lane) (n = 3) stained with Coomassie brilliant blue. (f) Western blot analysis (60 μg protein/lane) of EV preparations and complete SP using antibodies against EV-associated proteins CD9, CD63, and HSP70, endoplasmic reticulum marker calnexin (CNX), and a non-vesicular extracellular particle apolipoprotein C-III (ApoC3) (n = 3) (full western blot images in Supplementary Figure 2). SP: seminal plasma; P1–2: pool of fractions in size-exclusion chromatography; CL: cell lysate; Mw: molecular weight marker. Different letters (a, b) above the bars indicate statistically significant differences (p < 0.05) between methods (C, P1, and P2), as determined by a linear mixed-effects model and Tukey-adjusted pairwise comparisons. A significant positive correlation was observed between particle concentration and protein concentration across samples (Spearman’s ρ = 0.747, p = 0.0009). All data are expressed as mean ± SD of four independent experiments (n = 4). Scale bar = 200 nm.

Journal: Bio-protocol

Article Title: Uptake Assay of Ram Seminal Plasma Extracellular Vesicles to Sperm

doi: 10.21769/BioProtoc.5653

Figure Lengend Snippet: Analysis of SP-EVs fractions by (a) transmission electron microscopy (TEM) and (b, c) nanoparticle tracking analysis (NTA, n = 4). (d) Protein concentration (μg/μL) (n = 9) was assessed by Abs280 nm. (e) SDS-PAGE (15%) protein profile of SP-EV preparations and SP (60 μg protein/lane) (n = 3) stained with Coomassie brilliant blue. (f) Western blot analysis (60 μg protein/lane) of EV preparations and complete SP using antibodies against EV-associated proteins CD9, CD63, and HSP70, endoplasmic reticulum marker calnexin (CNX), and a non-vesicular extracellular particle apolipoprotein C-III (ApoC3) (n = 3) (full western blot images in Supplementary Figure 2). SP: seminal plasma; P1–2: pool of fractions in size-exclusion chromatography; CL: cell lysate; Mw: molecular weight marker. Different letters (a, b) above the bars indicate statistically significant differences (p < 0.05) between methods (C, P1, and P2), as determined by a linear mixed-effects model and Tukey-adjusted pairwise comparisons. A significant positive correlation was observed between particle concentration and protein concentration across samples (Spearman’s ρ = 0.747, p = 0.0009). All data are expressed as mean ± SD of four independent experiments (n = 4). Scale bar = 200 nm.

Article Snippet: 10% and 15% SDS-PAGE pre-cast gels (Bio-Rad, catalog number: 456-8033) 8.

Techniques: Transmission Assay, Electron Microscopy, Protein Concentration, SDS Page, Staining, Western Blot, Marker, Clinical Proteomics, Size-exclusion Chromatography, Molecular Weight, Concentration Assay