Criterion tgx stain free protein gel

Cells were lysed in Radioimmunoprecipitation assay (RIPA) lysis buffer. After lysis, the supernatant was collected. Equal amounts of protein (20 µg-40 µg) were run for 1.5 hours at 130V on a 4–20% Criterion TGX Stain-Free Protein Gel (Bio-Rad, Hercules, CA) and electrotransferred to a PVDF membrane by Iblot2 transfer stacks (Invitrogen, Waltham, MA). Transferred proteins were probed with both a Fli-1 polyclonal antibody described previously (20 (link)) and an antibody to β-actin (Cell Signaling, Beverly, MA). The results were visualized using the Odyssey Imaging System (LI-COR, Lincoln, NE).

Thirty micrograms of whole cell lysates were reduced in a mixture of 1x LDS sample Buffer (TruPAGE, PCG3009) and 50 μM DTT at 80°C for 10 minutes. Reduced lysates were then ran on a 4–15% Criterion TGX Stain-Free Protein Gel (BioRad, 5678083) in 1x tris/glysine/SDS (BioRad, 1610732) at 150 v for 1 hour and transferred to a nitrocellulose membrane in 1x tris/glycine (BioRad, 1610734) containing 20% methanol for 1 hour. The membrane was then washed in 1x TBS-T and blocked for 1 hour in 5% BSA-TBS-T. Primary mouse anti-IκBα (Novus, NB100-56507), mouse anti-phospho-IκBα S32/36 (Cell Signaling Technology, 9246S), and mouse anti-GAPDH (Biolegend, 607902) was then diluted to 1 μg/mL in 5% BSA-TBS-T and incubated at 4°C overnight with agitation. The membrane was then washed 3x for 10 minutes per wash in 1x TBS-T and secondary goat anti-mouse HRP (Abcam, ab205719) diluted 1:1000 in 5% BSA-TBS-T was incubated at room temperature for 1 hour after which the membrane was washed 3x for 10 minutes per wash in 1x TBS-T and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, 34094) on a BioRad ChemiDoc MP Imaging System.

The dynamic proteolysis of β-lg and lysozyme monomer and amyloid fibrils during digestion were analyzed by using 4–20% stain-free precast gels (Criterion TGX Stain-Free Protein Gel, Biorad Laboratories). The digested samples were diluted with a premade Laemmli sample buffer system including 0.065 M Tris-HCl with pH 6.8, 20 mg/ml SDS, 330 mg/ml glycerol, 0.01% (w/v) bromophenol blue, after which 50 μl β-mercaptoethanol was added and heated at 90 °C for 10 min. 15 μl of diluted sample was loaded into each well, and a voltage of 200 V was applied with the running buffer solution including 0.025 M Tris-HCl, 0.192 M glycine, and 0.1% (w/v) SDS. The images were obtained by scanning the gels with a molecular imager (ChemiDoc MP Imaging System, Bio-Rad Laboratories).

PHA blasts were lysed at a concentration of 2 × 10⁶ cells in 50 µl E1A lysis buffer (1% NP40, 20 mM HEPES, pH 7.9, 250 mM NaCl, 1 mM EDTA) complemented with protease inhibitors (Complete-ULTRA; 05 892 970 001; Roche). Prior to SDS-PAGE, samples were spun at 12,000 × g to remove insoluble material and were resuspended in 14 μl of loading dye. Equal amounts of protein (30 µg) were separated on a 4–15% agarose gel (Criterion TGX Stain-Free Protein Gel; Bio-Rad; 5678084) followed by semi-dry transfer to nitrocellulose. Proteins were visualized by chemiluminescence (SuperSignal West Femto; ThermoFisher; 34094). Antibodies used recognize Roquin-1/2 (Millipore; 3F12), CNOT1 (Proteintech; 14276-1-AP), Edc4 (Cell Signaling Technology; 2548), β-Tubulin (Abcam; ab21058), and β-Actin (Santa Cruz; c4, sc-47778). All uncropped images can be retrieved in the Source Data file.

Stain-free 4%-20% SDS PAGE (Criterion TGX Stain-Free Protein Gel; #5678094; BioRad, Hercules, CA) and Western Blot analysis were used to characterize the purified IgY. The two primary antibodies used were: rat anti-IgY (Sapphire, LO-IgY-16, batch: 10332, Ann Arbor, Michigan), diluted 1:500 and mouse anti-ovalbumin (Abcam; ab17293, lot number: GR3376118; Cambridge, MA) diluted 1:500, followed by goat anti-rat HRP (EMD Millipore; AP136P, Lot. Number: 3660343; Billerica MA) Cat. No: NA931V (Rock Immunochemicals; RL610603002, Lot. No: 17041904; Pottstown, PA) and sheep anti-mouse HRP, respectively.

Purified, recombinant Bmh1 and Hst2 isoforms (0.3 mg each) were mixed with 20-μl 50:50 slurry of Avidin Agarose Resin (Fisher Scientific) in 80 μl of Bmh1 size exclusion chromatography buffer (20-mM Tris, pH 8, 100-mM NaCl, 0.5-mM TCEP, 10% glycerol), incubated for 1 h at RT with shaking (300 rpm) and subsequently washed three times with PBS. Proteins were eluted by boiling in the SDS-PAGE sample buffer, analyzed by 10% SDS-PAGE, and stained with InstantBlue.
For H3 peptide pull-down reactions, 5-mg peptide was coupled to N-hydroxysuccinimide-activated Sepharose 4B (GE Healthcare Life Sciences). To prepare unphosphorylated H3S10 peptide for control reactions, beads were incubated with CIP at 37 °C for 1 h.
Purified, recombinant Bmh1 and unmodified or phosphorylated Hst2 isoforms (0.3 mg each) were mixed with 20-μl peptide-coupled beads in 80 μl of Bmh1 size exclusion chromatography buffer (20-mM Tris, pH 8, 100-mM NaCl, 0.5-mM TCEP, 10% glycerol), incubated for 1 h at RT with shaking (300 rpm) and subsequently washed three times with PBS. Proteins were eluted by boiling in the SDS-PAGE sample buffer, analyzed by 4 to 15% Criterion TGX Stain-Free Protein Gel (Bio-Rad) and stained with InstantBlue.

HPLC-SEC was used to determine antibody concentration and purity, using a TSK_gel G3000SW_XL column (7.8 mm i.d. x 30 cm; Tosoh Bioscience). 100 µL of sample was injected for analysis, utilising a flow rate of 0.6 mL/min and a mobile phase which consisted of 0.2 M L-arginine, 0.05 M MES, 5 mM EDTA, 0.05% sodium azide (w/w), pH 6.5. The resultant concentrations were obtained by comparing the area under the peaks obtained at UV absorbance 280 nm with that of a calibration curve obtained using standard samples. The relative amount of HMW and LMW species was calculated based on the area of elution peaks before and after the monomeric peak, respectively. The area of the respective species obtained from HPLC-SEC was multiplied with the respective volume obtained from the AKTA system in order to perform mass balance analysis. Non-reducing SDS-PAGE gels (4–15% Criterion™ TGX Stain-Free™ Protein Gel, Bio-rad) were used according to manufacturer’s instructions, as a complementary approach to investigate the purity of the samples. Staining was performed with eLuminol™ (GeneCopoeia), with a total protein amount of 0.3 µg loaded per lane, as determined using Bradford assay (Thermo Fisher Scientific).