Equivalent amounts of total cell lysates collected from B. burgdorferi cultures (1 – 3 × 107 cells ml−1) were analyzed by SDS-PAGE using pre-cast Novex 4–20% Tris-Glycine polyacrylamide gels (Invitrogen) and transferred to PVDF Immobilon membranes (Millipore) as previously described (Drecktrah et al., 2013 (link)). Protein levels were analyzed by incubating membranes with rabbit antibodies against OspC (1:1000) or mouse antibodies against FlaB (1:50) followed by goat anti-rabbit or goat anti-mouse HRP-linked antibodies (Bio-Rad Laboratories) (1:5000). Blots were developed by chemiluminescence (Amersham ECL Prime, GE Healthcare) and visualized using an LAS-3000 Intelligent Dark Box (Fujifilm Medical Systems USA).
Protocol detail
SDS-PAGE and Western Blot Analysis of B. burgdorferi
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Novex 4–20% Tris-Glycine polyacrylamide gels
PVDF Immobilon membranes
goat anti-rabbit or goat anti-mouse HRP-linked antibodies
Amersham ECL Prime
LAS-3000 Intelligent Dark Box
Anti antibodies
Antibodies
Cast
Cells
Chemiluminescence
Glycine
Goat
Immobilon
Membranes
Mouse
Ospc
Polyacrylamide gels
Protein
Pvdf
Rabbit
Sds page
Tris
Corresponding Organization : University of Montana
Other organizations : University of Wisconsin–Madison
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Variable analysis
independent variables
- Equivalent amounts of total cell lysates collected from B. burgdorferi cultures (1 – 3 × 10^7 cells ml^−1)
- Protein levels of OspC and FlaB
- Equivalent amounts of total cell lysates
- SDS-PAGE using pre-cast Novex 4–20% Tris-Glycine polyacrylamide gels
- Transfer to PVDF Immobilon membranes
- Incubation of membranes with rabbit antibodies against OspC (1:1000) or mouse antibodies against FlaB (1:50)
- Incubation with goat anti-rabbit or goat anti-mouse HRP-linked antibodies (1:5000)
- Blot development by chemiluminescence (Amersham ECL Prime, GE Healthcare)
- Visualization using an LAS-3000 Intelligent Dark Box (Fujifilm Medical Systems USA)
- Not explicitly mentioned
- Not explicitly mentioned
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