Western Blot Analysis of Sea Urchin Immune Proteins

Bacteria with bound SpTrf proteins or samples of the nickel column eluates were heated to 95°C in SDS lysis buffer and separated by electrophoresis on a 12% sodium dodecyl sulfate—polyacrylamide gel (SDS-PAGE), transferred to PVDF membranes, and evaluated with anti-SpTrf antibodies according to [46 (link), 49 (link)]. The sea urchin complement C3 homologue, SpC3, was detected using anti-SpC3-6H as described [53 (link), 64 (link), 65 (link)]. The antibody against SpC3 was raised to a 50 kDa recombinant fragment of the N terminal region of the SpC3 α chain [64 (link)]. Secondary antibodies were labeled with horse radish peroxidase, and blots were processed for ECL chemiluminescence (Thermo Fisher Scientific) followed by imaging with a ChemiDoc XRS+ (Bio-Rad Laboratories).

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Chou H.Y., Lun C.M, & Smith L.C. (2018). SpTransformer proteins from the purple sea urchin opsonize bacteria, augment phagocytosis, and retard bacterial growth. PLoS ONE, 13(5), e0196890.

Publication 2018

ECL chemiluminescence ChemiDoc XRS+

Anti antibodies Antibodies Antibody Bacteria proteins Buffer Chemiluminescence Complement c3 Electrophoresis Horse radish peroxidase Membranes Nickel Polyacrylamide gel Pvdf Sds page Sea urchin Sodium dodecyl sulfate

Corresponding Organization : George Washington University

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Variable analysis

independent variables

Heating bacteria with bound SpTrf proteins or samples of the nickel column eluates to 95°C in SDS lysis buffer

dependent variables

Detection of the sea urchin complement C3 homologue, SpC3, using anti-SpC3-6H antibody
Detection of SpTrf proteins using anti-SpTrf antibodies

control variables

Separation of heated samples by electrophoresis on a 12% sodium dodecyl sulfate—polyacrylamide gel (SDS-PAGE)
Transfer of separated proteins to PVDF membranes
Use of secondary antibodies labeled with horse radish peroxidase
Processing of blots for ECL chemiluminescence and imaging with a ChemiDoc XRS+ (Bio-Rad Laboratories)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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