For isoelectrofocusing (IEF), 140 μL of sample solution containing around 30 μg total protein, 18.2 mM dithiotreitol (DTT), and 0.5% immobilized pH gradient buffer solution were loaded into 7 cm non linear pH 3 10 strips.
After cross-validation of the biosensing performance of the LSPR platform with the ELISA technique, THP-1 cells suspended in buffer solution were loaded into the LSPR device for on-chip cell separation, incubation, stimulation, and detection of cell-secreted TNF-α.
Free CTX at 500, 250, 125, and 62.5 μg/mL was mixed with an equal volume of Laemmli Sample Buffer and the solution was loaded (30 μL) in the precast gel as standards.
To separate free dye from the scFv-bound dye and to exchange the buffer to PBS, the solution was loaded on a microcon concentrator column YM-10 (Millipore) with a membrane-cutoff of 10 kDa.
0.8 g free BCL powder was dissolved in the 5 mL 0.05 M phosphate buffer (pH 7), this solution was loaded into the tube to mix with the MPR.
After 1 10 dilution in 50 mM sodium phosphate loading buffer pH 7.0, the solution was loaded on a 1 mL HiTrap-HIS column (GE Healthcare) that was previously loaded with 1 mL 0.5 M NiSO4 and washed with 10 column volumes of loading buffer.
Pellets were resuspended in 20 µl of 1× NuPage SDS reducing buffer and 10 µl of each solution was loaded on (12%) NuPAGE Novex Bis-Tris Gels 12 wells (Invitrogen, USA) for electrophoresis under constant application of 120 V for 45 min. Electroblotting was performed onto polyvinylidene fluoride membranes (150 V for 1 h).
Sample buffer (Beyotime, China) was added to the cytoplasmic protein and the solution was loaded onto a 6% polyacrylamide gel.
Then, a solution made from 2.0 mL of phosphate buffer (0.2 M, pH 7.4) and 400 μL of the 100 μg/mL KA solution was loaded into a quartz cell.
Subsequently, the protein solution was loaded onto a HiTrap Q FF prepacked with Q Sepharose (GE Healthcare Life Sciences) equilibrated with Buffer A and the bound proteins eluted using a buffer elution gradient from 0 to 1000 mM of NaCl.
