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01 Lyse cells in cold RIPA 02 Resolve on 4–12% gel 03 Wet-transfer to PVDF…
Journal of Negative & Applied Results
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Western blot v3
Western blot for membrane proteins
AM
A. Moreau ORCID 0000-0002-1825-0097
FIX HISTORY
v3integrated
Cold wet-transfer at 4 °C — fixed stripping of the ATPase band.
v2negative result
5% milk blocked the signal — recorded, switched to BSA.
v1accepted
Initial publication — structured steps & reagents.
Reproducibility: healthy
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membrane-protein-western-blot.pdf
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Detected 6 steps4 reagents2 figures
Western blot for membrane proteins
v1 · draft Reagents
RIPA bufferPVDF membraneAnti-Na⁺/K⁺ ATPaseTris-glycine 4–12%
Steps
01 Lyse cells in cold RIPA with protease inhibitors; avoid boiling membrane proteins — incubate at 37 °C for 30 min.
02 Resolve 20 µg lysate on a 4–12% Tris-glycine gel at 120 V.
03 Wet-transfer to PVDF, 100 V for 90 min at 4 °C.
04 Block 1 h in 5% milk/TBST; probe with primary antibody overnight at 4 °C.
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6 steps · 4 reagents · 2 figures extracted
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replication negative result pending review
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− Block in 5% milk/TBST
+ Block in 5% BSA/TBST
v2 → v3 · by R. Tanaka
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