Journal: Animals : an Open Access Journal from MDPI

Article Title: Binding of Equine Seminal Lactoferrin/Superoxide Dismutase (SOD-3) Complex Is Biased towards Dead Spermatozoa

doi: 10.3390/ani13010052

Figure Lengend Snippet: ( a ) SDS-PAGE of multiple attempts to isolate lactoferrin (80 kDa) from seminal plasma (lanes 1–3). A second protein at 32 kDa consistently co-eluted with lactoferrin. Lane 4: Recombinant human lactoferrin. ( b,c ): Western Blots of SOD-3 LF. ( b ) Western blot using anti-SOD-3 antibody (1:1000, SCBT, sc-67088) identified the second protein as SOD-3. Lanes 1–3: Lactoferrin purified using different methods. Lane 4: Recombinant human lactoferrin. ( c ) Western Blot using anti-lactoferrin antibody (1:1000, SCBT, sc-14434) of purified lactoferrin (lane 1).

Article Snippet: Protein distribution and purity of the isolated fractions were determined by SDS-PAGE and by Western blot using goat anti-human lactoferrin polyclonal antibody (1:1000, sc-14434, Santa Cruz Biotechnology, Inc., Santa Cruz; no longer in production) and mouse SOD-3 monoclonal antibody (1:1000, sc-58427, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: SDS Page, Clinical Proteomics, Recombinant, Western Blot, Purification

Journal: Redox Biology

Article Title: Redox regulation of nitrosyl-hemoglobin in human erythrocytes

doi: 10.1016/j.redox.2019.101399

Figure Lengend Snippet: Antioxidant enzymatic systems in human RBCs. A) Representative Western blot analysis of antioxidant enzymes expressed in the cytosol of human RBCs. Human total erythrocyte lysates, cytosolic fractions and isolated ghost membrane samples (20 μg of proteins each) were analyzed by immunoblotting analysis as described in Materials and Methods; bands specific for SOD1 (at 17 kDa); Catalase (at 60 kDa); GPX1, Prdx1 and 2 (at 22 kDa); and Prdx6 (at 26 kDa) are shown using chemiluminescent HRP-based detection. HUVEC cell lysates were used as positive control (except catalase). B) Representative image of immunofluorescent detection of SOD3 (upper-right panel) and CD235a (or Glycophorin A; lower-left panel) in healthy human RBCs as described in Materials and Methods. The upper-left panel shows the overlay of both immunodetected signals. As negative control (bottom-right panel), RBCs were stained with conjugated secondary antibody only (Alexa Fluor-488 anti-mouse IgG).

Article Snippet: Erythrocytes were immunoblotted, after extensive washing, with an anti-SOD3 (sc-67089, Santa Cruz) and anti-CD235a (555569, BD) antibodies for all night at 4 °C, and subsequently washed and incubated with secondary antibodies Alexa-488 goat anti-rabbit and Alexa-568 anti-mouse (Invitrogen) for 1 h. The preparations were mounted in fluorescent counting medium (DAKO) and examined in a fluorescence microscope equipped with an ApoTome module for structured illumination (Zeiss AxioImager.z1, USA).

Techniques: Western Blot, Isolation, Positive Control, Negative Control, Staining