After separation by SDS-PAGE or native-PAGE (see above) the proteins were transferred onto PVDF membranes. The membranes were blocked with 5% (w/v) dried skimmed milk in TBS (25 mM Tris-HCl pH 7.5, 150 mM NaCl) and incubated with primary antibodies followed by IRDye fluorescently labeled secondary antibodies (LiCor). The membranes were scanned with an Odyssey near-infrared imager (LiCor). Primary antibodies and antisera against hamster BiP [chicken anti-BiP; 33 (link)], eIF2α [mouse anti-eIF2α; 34 (link)], and FICD [chicken anti-FICD 12 (link)] were used.
Protocol detail
Quantitative Western Blot Analysis
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Antibodies
Antisera
Chicken
Hamster
Membranes
Milk
Mouse
Nacl
Native page
Proteins
Pvdf
Sds page
Tris
Corresponding Organization : University of Cambridge
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Variable analysis
independent variables
- SDS-PAGE or native-PAGE separation method
- Primary antibodies used: chicken anti-BiP, mouse anti-eIF2α, chicken anti-FICD
- Protein expression levels of BiP, eIF2α, and FICD
- Blocking of membranes with 5% (w/v) dried skimmed milk in TBS (25 mM Tris-HCl pH 7.5, 150 mM NaCl)
- Incubation with IRDye fluorescently labeled secondary antibodies
- Scanning of membranes with an Odyssey near-infrared imager (LiCor)
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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