Criterion blotter transfer tank

To avoid bias in protein levels due to possible plasma protein contamination, the PBMC samples were normalized by cell number [35 (link)]. Aliquots of 25 μL (5 ×10⁵ cells) were loaded per well onto 4-15% TGX Criterion^™ 18- well precast gels in a Criterion^™ vertical midi-format electrophoresis cell (Bio-Rad Laboratories, Hercules, CA), containing 0.5 L/gel of 1x Tris/Glycine/SDS running buffer. Additionally, 7 μL aliquots of a commercially available mixture of prestained protein standards were loaded into the first, middle, and last well. Proteins were fractionated under a constant 200 V applied for ~40 min. Upon SDS-PAGE completion, gels were equilibrated in cold Towbin buffer for 10 min. Resolved proteins were electrophoretically transferred out of the PAGE gel and onto 0.45 μm Immobilon-FL polyvinylidene difluoride (PVDF) transfer membranes (Millipore, Billerica, MA) with 100 V applied for 1.5 h in a Criterion blotter transfer tank (Bio-Rad Laboratories, Hercules, CA) filled with cold Towbin buffer. To maintain chilled transfer conditions, the tank's ice packs were changed at 45 min intervals.

Protein extraction was performed with the AllPrep® DNA/RNA/Protein Mini Kit (QIAGEN) according to the manufacturer’s instructions. Proteins were separated on Criterion Midi Gels (Bio-Rad Laboratories) in 25 mM Tris base (Univar), 190 mM glycine (Univar) and 0.1% sodium dodecyl sulphate (Sigma-Aldrich) in MilliQ water, pH 8.3 buffer. Proteins were transferred onto nitrocellulose membranes (GE Protran) using the Criterion Blotter transfer tank (Bio-Rad Laboratories) in a 25 mM Tris base, 190 mM glycine, and 20% methanol (Univar) buffer. The membranes were incubated with the following primary antibodies diluted in 5% bovine serum albumin (BSA) (Life Technologies) in Tris base sodium chloride (Univar) with 0.1% Tween-20 (Sigma-Aldrich) (TBST) and incubated overnight at 4 °C: anti-P glycoprotein (ABCB1) antibody ([EPR10364-57], Abcam, Cat#ab170904) at 1:500, and GAPDH antibody (Santa Cruz Biotechnology, Cat#sc-47724) at 1:1000. Secondary goat anti-rabbit (Life Technologies, Cat# 32460) and goat anti-mouse (Life Technologies, Cat#32430) were diluted 1:1000 in 5% BSA in TBST and incubated at room temperature for 1 h. Signals were visualised and quantified on the Bio-Rad ChemiDoc Touch System (Bio-Rad Laboratories) using Image Lab software v5.2.1 (Bio-Rad Laboratories).^{37 (link)}