Coomassie brilliant blue r 250 staining solution

CMG2-Fc standard from Planet Biotechnology (0.5, 0.75, 1.0, 1.25, and 1.5 μg/lane) and rCMG2-Fc variants (APO, ER, and Agly) were reduced, denatured, and run on a 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA) gel at 50 mA for 1.5 h. After staining for 1 h with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad Laboratories, Hercules, CA), the gel was washed with water overnight. Next morning, the gel was scanned with Gel Doc™ XR+ System (Bio-Rad laboratories, Hercules, CA), and a standard curve was established by plotting total protein mass of standards as a function of band intensity. Then, the band intensity for the ~50 kDa band of rCMG2-Fc variants was interpolated onto the standard curve to determine the mass of intact rCMG2-Fc, and calculate their concentrations.

To determine whether the soluble proteins quantified encompass proteins of various molecular weights, especially the low molecular weight proteins or less abundant proteins which were eradicated in harsh procedures, protein visualization was conducted using Coomassie Blue staining. 50 μL of each soluble protein was mixed with 50 μL of 2x Laemmli sample buffer (Biorad, cat # 161-0737) and heated for 10 min at 100 °C. Samples were stored at −80 °C or kept on ice while working. SDS-polyacrylamide gel 12% was prepared as described by Harlow and Lane (Harlow & Lane, 1988 ). 20 μg of every sample’s protein was loaded in a well. The gel was allowed to run for 10 minutes at 100 V then the voltage was raised to 200 V for 20–30 minutes. Gels were fixed by submerging in fixing solution (50% water; 40% Ethanol; 10% acetic acid) for 10–30 minutes, then washed in distilled water for 10 minutes, and finally stained with Coomassie Brilliant Blue R-250 staining solution (Biorad, cat # 161-0436) for 1–4 hours. To enhance visualization of bands, the gels were de-stained with de-staining solution (50% methanol, 5% acetic acid, 45% water) for 45 minutes.

From the obtained protein sample, 20 μg of protein was mixed with 1x Laemmli sample buffer (Bio-Rad, Munich, Germany), denatured for 5 min at 95 °C, and separated by using 10% SDS-PAGE at 200 V for 25 min. The gel was incubated in a fixation solution composed of 50% ethanol and 10% acetic acid in ddH₂O for 30 min at RT. Then, the gel was stained with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad) for 10 min at RT, destained in 7.5% acetic acid and 25% methanol in ddH₂O for 1 h at RT and stored in ddH₂O overnight. Afterwards, a single band at around 42 kDa and additionally all bands from around 25 to 100 kDa were cut out and used for MS analysis.

Protein purity estimation was carried out using SDS-PAGE on 4–20% precast acrylamide gels (Biorad 4–20% Mini-Protean TGX Precast Protein Gels, 15-well). Gels were stained with Coomassie Brilliant Blue R-250 Staining solution (Biorad).
Transferring proteins from SDS-gels to PVDF membranes (BioRad) was performed using the BioRad Turbo blotting system (Trans-Blot Turbo Transfer System). After the transfer, membranes were washed three times with PBS-T (PBS, 0.08% Tween-20, pH 7.4) and blocked with 5% BSA in PBS-T. Afterwards, membranes were incubated with the primary anti-DPP3 mAb (murine AK1967, 2 μg/mL in PBS-T with 1% BSA) over night at 4°C on a shacking platform and washed five times. Membranes were incubated with secondary antibody (Anti-mouse IgG, HRP-linked antibody, Cell Signaling Technology, 1:5000 in PBS-T with 1% BSA) at room temperature for 1 hour on a rocking shaker. Afterwards membranes were washed three times. Membranes were developed by incubation with ECL Prime Western Blotting Detection Reagents (Amersham GE Healthcare). Chemiluminescence was detected using the LAS 500 imager system (GE Healthcare).

MMP-9 activity in the conditioned medium was detected through gelatin zymography. The sample was examined under nonreducing conditions by using an 8% sodium dodecyl sulfate (SDS) polyacrylamide gel containing 1 mg/mL gelatin. The gel was washed in 2.5% Triton X-100 to remove SDS and to allow MMP renaturation. Moreover, the gel was incubated with reaction buffer (0.2 M NaCl, 50 mM Tris [pH 7.5], 5 mM CaCl₂, and 0.02% BRIJ^® 35) at 37 °C for 48 h, after which it was stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad Laboratories, Hercules, CA, USA). The presence of pro-MMPs and active MMPs resulted in white lysis bands attributable to gelatin degradation.