Any kd mini protean tgx stain free protein gel

Cladophora cellulose was provided by FMC Biopolymer (batch 3095-10; Newark, DE, USA). FIX-rich PCC was provided by National Center for Hematology, Moscow, Russia, as lyophilized powder. Coliphages ΦX174 (ATCC 13706-B1™) and PR772 (BAA-769-B1), and the host bacteria Escherichia coli (Migula) Castellani and Chalmers C (ATCC 13706) and K12 J53-1(R15) [HER 1221] (BAA-769) strains were obtained from ATCC (Manassas, VA, USA). Agar (214530) was obtained from BD (Franklin Lakes, NJ, USA). Tryptone (LP0042B) and yeast extract (Oxoid) (LP0021) were obtained from Thermo Fisher Scientific. Phosphate-buffered saline (P4417), 2-mercaptoethanol (M3148), sodium chloride (S5886), sodium phosphate dibasic (71640) and 2-mercaptoethanol (M3148) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Any kD™ Mini-PROTEAN^® TGX Stain-Free™ protein gels (4568125), tris/glycine/SDS running buffer (1610732), 4x Laemmli Sample Buffer (1610747), and Precision Plus Protein™ unstained protein standards (1610363) were purchased from Bio-Rad (Hercules, CA, USA).

A total of 0.7 m BON and 1 m NCI-295R cells per well were seeded in 6-well plates and treated for 24 h with 0.1 µg/mL TNFα starting the following day. Cells were lysed in RIPA buffer containing Complete Mini Protease Inhibitor Cocktail (Roche). The protein concentration was measured using the BCA kit (Thermo Fisher) with the Tecan Sunrise plate reader (Tecan, Männedorf, Switzerland). A sample with 15 µg of proteins was loaded on Any kD™ Mini-PROTEAN^® TGX Stain-Free™ Protein Gels (Bio-Rad, Hercules, CA, USA) and run at constant 100 V until the dye front reached the reference line of the gel. For protein transfer, PVDF membranes and Trans-Blot Semi-Dry Transfer Cell (Bio-Rad) were used. Membranes were cut and after blocking with Blotting-Grade Blocker (Bio-Rad), the upper halves were incubated with mouse anti-MVP antibody (Abcam, Cambridge, UK) and the lower halves were incubated with mouse β-actin antibody (Sigma-Aldrich) at +4 °C overnight, washed, and incubated with HRP-labeled anti-mouse antibody (GE Healthcare). As the ECL substrate, Western Lightning Plus (PerkinElmer, Waltham, MA, USA) was used. The luminescence signal was captured with the Hyperfilm ECL (GE Healthcare).

Samples for SDS–PAGE analysis of the purification progress were prepared using 2× Laemmli sample buffer (Bio-Rad). Samples for analysis via SDS–PAGE followed by western immunoblotting were loaded onto a 4–12% Bis–Tris NuPAGE gel (Novex), along with Sharp Pre-stained protein standard (Novex) and run at 200V in 1× MES SDS running buffer (NuPAGE). Samples for SDS–PAGE analysis were loaded onto an any-kD Mini Protean TGX Stain-free protein gel (Bio-Rad), along with Precision plus unstained marker (Bio-Rad) and run at 300 V in 1× Tris/Glycine/SDS buffer (Bio-Rad). any-kD Mini Protean TGX Stain-free protein gels were visualized using the Chemidoc MP system (Bio-Rad). The loaded sample volume ranged between 15 and 20 µl.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed according to the method of Laemmli [42 (link)], as follows. Each culture supernatant or purified protein was mixed with reduced Laemmli sample buffer containing dithiothreitol (DTT; final concentration 200 mM) and incubated at 100 °C for 3 min. Each sample was loaded on an Any kD™ Mini-PROTEAN^® TGX Stain-Free™ Protein Gel (Bio-Rad, Hercules, CA, USA) and subjected to electrophoretic separation. Chemifluorescent signals were captured using a Chemi Doc MP Imaging system (Bio-Rad). Precision Plus Protein Unstained Standards (Bio-Rad) were used as molecular weight markers. The protein bands were analyzed using Image Lab software version 4.0 (Bio-rad). The protein level of mature BLP in each culture supernatant was calculated from the intensity of the band at the position of mature BLP (19 kDa), using the serial diluent of purified wild-type BLP (quantified by DC-protein assay kit (Bio-Rad)) as the standard. Non-reducing SDS-PAGE was performed in the same way as above, but using DTT-free Laemmli sample buffer instead of the reduced buffer.

The scGFP constructs were transformed into E. coli TOP10 cells (Invitrogen) and transformants were inoculated in 100 ml LB medium containing 50 μg/ml ampicillin and grown to 0.6 OD₆₀₀ with shaking at 37 °C. After addition of 0.2% L-arabinose, the cultures were transferred to 16 °C for 12–16 h, harvested by centrifugation and resuspended in 1 ml of 20 mM Tris pH 7.4, 1 M NaCl, 1 mM TCEP, 5 mM MgCl₂, 5 mg/ml lysozyme, 20 μg/ml DNase I and protease inhibitor cocktail (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 5 μg/ml antipain, 157 μg/ml benzamidine) followed by 3 rounds of freeze-thawing and 3 rounds of sonication. The lysates were centrifuged at 20,000 × g for 30 min at 4 °C and the supernatant was used for native gel analysis. To this end, 37.5 μl supernatant was mixed with 12.5 μl 4× DNA/Native sample buffer (Expedeon) and loaded onto an Any kD™ Mini-PROTEAN^® TGX Stain-Free™ Protein Gel (BIO-RAD) after normalization of samples for total GFP fluorescence. The gel was run in 27.3 mM Tris-HCl and 192 mM glycine at 4 °C and visualized using a Typhoon FLA 9500 Biomolecular Imager (GE Healthcare) with 473 nm excitation laser and BPB1 emission filter (530/30).