Pierce coomassie bradford protein assay

Total protein was extracted in RIPA buffer (1% Igepal, 0.1% SDS, 0.5% sodium deoxycholate, 0.05 M Tris HCl pH 7.4; all Sigma-Aldrich) supplemented with complete protease inhibitor and phosphatase inhibitor PhosSTOP (both Roche). Protein concentration was quantified using Pierce Coomassie (Bradford) Protein Assay (Thermo Fischer Scientific). TGX stain-free 4–20% gradient gels and Trans­Blot Turbo™ PVDF membranes (both Bio-Rad) were used for western blotting. Specific proteins were detected with the following primary antibodies: β-actin (Sigma-Aldrich, A5316), β-tubulin (Cell Signaling Technologies (CST), 2128), CDK2 (SC, sc-163), CDK4 (CST, 2906), CDK6 (Abcam, ab124821), cyclin D1 (CST, 92G2), cyclin E1 (CST, 4129), cyclin E2 (CST, 4132), ER (Thermo Fisher Scientific, SP1), retinoblastoma protein (Rb) (SC, sc-102), phospho-Rb (Ser807/811) (CST, 8516), and phospho-Rb (Thr821) (Abcam, ab4787) followed by HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Thermo Fisher Scientific). Signals were detected using Luminata Forte Western HRP Substrate (Merck-Millipore) and a ChemiDoc MP (Bio-Rad). All bands were normalized to total lane protein employing stain-free technology and quantified using the Image Lab™ Software v5.2.1 (Bio-Rad). For clarity, β-actin/β-tubulin loading controls are presented for all western blots.

IL-6 and Il-1β levels were determined in the supernatant by western blotting. The supernatant was collected after treatment and protein was precipitated using 4 volumes of ice-cold acetone. The protein was dissolved in RIPA buffer containing protease inhibitors before determining the concentration of cellular protein using the Pierce Coomassie Bradford protein assay (Thermo Fisher Scientific, Cat. #23236). Samples were separated on a 4-15% gel before being transferred to a PVDF membrane to probe for IL-1β (1:500), IL-6 (1:500), and β-actin (1:2000) as previously described (27 (link)). Blots were incubated with primary antibodies overnight at 4°C before being treated the following day with anti-rabbit secondary horse radish peroxidase-conjugated antibodies (1:10,000 for 1 hr at room temperature; Abcam, Cambridge, UK). Membranes were washed, incubated with a Luminata Forte Chemiluminescence (Millipore) for protein detection, and analyzed using an ImageQuant LAS 4000 imager and software (GE Healthcare Life Sciences, Marlborough, MA).

Trypsin enzyme activity was measured in the pancreas and in cecal homogenates (1:10 wt/vol). The pancreatic homogenates were incubated with enteropeptidase to activate trypsin before incubating with a trypsin-specific substrate, benzoyl-DL-arginine-4-nitroanilide (BAPNA; Merck, Merck Life Science AB, Darmstadt, Germany f.k.a Sigma-Aldrich). The trypsin activity unit (U) was recalculated as the amount of enzyme that catalyzes 1 μmol of substrate per minute.
A universal protease activity assay was performed to estimate the total proteolytic activity in freshly squeezed juice made from pineapple or papaya fruit for dose recalculation and in murine cecal homogenates. In brief, samples were incubated with casein as a protease substrate at +37 °C for 10 min while the quantity of liberated amino acid tyrosine during this time was measured after reacting with Folin’s reagent according to manufacturer technical protocol (Merck, Merck Life Science AB, Darmstadt, Germany) [27 (link)]. L-tyrosine with known concentrations was used for standard curve preparation.
The protein concentration in the supernatants was determined using the Pierce Coomassie (Bradford) Protein assay with serum albumin as a standard (Thermo Fisher Scientific, Waltham, MA, USA). The activity of enzymes was recalculated per mg of total protein.