Rc dctm protein assay kit

The regenerating liver tissues stored in liquid nitrogen were ground into fine
powder and then suspended in extraction buffer (7 M urea, 2 M thiourea, 4%
CHAPS). Next, the suspension was vortex-mixed for 1 h at 4 °C, and subsequently
centrifuged at 20,000 x g for 1 h at 4 °C. The supernatants
were collected and stored at –80 °C for further use. The protein concentration
was assessed with the commercial RC DC^TM Protein Assay Kit according
to the manufacturer’s instructions (BIO-RAD, USA). Protein samples, 50 μg, were
separated by electrophoresis on 12% SDS/PAGE gels and subsequently
electrophoretically transferred to polyvinylidene difluoride membranes
(Millipore). The membranes were blocked with 5% non-fat milk, washed, and
subsequently probed with antibodies against 14-3-3β/α, γ, ζ/δ, σ, ε, η, τ/θ (all
1:1000) and GAPDH (1:2000) (Sangon Biotech Co. Ltd., Shanghai, China)overnight
at 4 °C. After being washed, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies (Sangon Biotech Co. Ltd., Shanghai,
China), detected with an enhanced chemiluminescence detection kit (Boster
Corporation, China) and then imaged in an ImageQuant LAS 4000 mini (GE
Healthcare Bio-Sciences Corporation) system.

Telomerase activity was measured using the telomere repeat application protocol (TRAP) assay with the TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche Diagnostics, Mannheim, Germany). This kit provides a way to perform a highly sensitive photometric enzyme immunoassay to detect “telomerase activity” [5 (link)]. We used 3 μg of total protein in the PCR reaction to measure telomerase activity and determined protein concentrations using an RC DC^TM protein assay kit (Bio-Rad, CA, USA).
Telomerase adds telomeric repeats (TTAGGG) to the 3’-end of the biotin-labeled synthetic P1-TS-primer. These elongation products, as well as the Internal Standard (IS) included in the same reaction vessel, are amplified by PCR using the primer P1-TS and the anchor-primer P2. The PCR products are split into two aliquots, denatured and hybridized separately to digoxigenin (DIG)-labeled detection probes specific for the telomeric repeats (P3-T) y. Immobilized amplicons are then detected with an antibody against horseradish peroxidase-conjugated digoxigenin (Anti-DIG-HRP) so that telomerase activity can be detected from PCR measurements.

Protein extraction from PBMC samples was performed using RNA/DNA/Protein Purification Plus Kit (Norgen Biotek, Ontario, Canada). Proteins were measured using the RC DCTM protein assay kit (Bio-Rad, USA) in Multiskan MS plate reader (Labsystems, Vantaa, Finland) and the concentration was calculated by plotting standard curve. Protein samples (20–60 μg) subjected to SDS-PAGE using 8% polyacrylamide gels and transferred to Amersham Hybond PVDF membranes by either wet or semi-dry transfer systems. The membranes were blocked with 10% FBS in Tris buffered saline containing 0.1% Tween (TBS-T) for 1 h and incubated overnight at 4°C with primary antibodies against STIM2 (1:200, Cell Signaling Technology, Cat No. 4917), ORAI1 (1:500, Alomone labs, Cat No. ACC-060), ORAI2 (1:500, Alomone labs, Cat No. ACC-061), Ca_V1.3 (1:500, Alomone labs, Cat No. ACC-005), Ca_V2.3 (1:500, Alomone labs, Cat No. ACC-006) and GAPDH (1:3000; Merck Millipore, Cat No. ABS16). After intensive washing with TBS-T, the membranes were further incubated with horseradish peroxidase-conjugated secondary antibody (1:3000; Cell Signaling Technology, Cat No. 7074) for 2 h. Further, membranes were washed intensively and developed for detection of immunoreactive protein bands using ECL Prime Western Blotting System (GE Healthcare, RPN2232). Bands were visualized in ChemiDoc^MP imaging system (Bio-Rad).