PBMCs were washed with PBS, and lysed with RIPA buffer (Solarbio, R0010), and quantified using a BCA assay kit (Beyotime, P0012) according to the manufacturer’s instructions. The samples were then boiled 5 minutes at 95 °C in a 5 × protein loading buffer (Meilunbio, MA0003-D). An equivalent quantity of protein samples was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Beyotime, P0012AC) and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, IPVH15150) using WB transfer buffer (Solarbio, D1060). After blocking non-specific binding with 5% skimmed milk (BioFroxx, 1172GR500), rabbit anti-STAT5 (phospho Y694) monoclonal antibody (Abcam, ab32364) and mouse β-actin primary antibody (Abcam, ab8226) were incubated overnight at 4°C. After washing with Tris-buffered solution (Solarbio, T1080) for three times, the blots were incubated with species-specific secondary antibodies (Abcam, ab6721/ab6728) for two hours at room temperature. After five additional washes with Tris-buffered solution containing 0.01% Tween 20, the protein bands were visualized using an ECL reagent kit (Solarbio, PE0010) based on the manufacturer’s instructions (Carestream, USA). Images were captured with a Chemiluminescence Imaging System (CLINX, ChemiScope 6100 Touch, China).
Chemiscope 6100 touch
Manufactured by Clinx
Sourced in China
The ChemiScope 6100 Touch is a laboratory imaging system that provides high-quality digital images of samples. It features a large 10-inch touchscreen display for intuitive operation and a high-resolution camera for capturing detailed visual data.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using chemiscope 6100 touch
1
STAT5 Phosphorylation Assay in PBMCs
2
Western Blot Analysis of Protein Samples
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Isolated islets or cell pellets were suspended in strong RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, and 50 mM Trizma base, pH 8.0) containing protease inhibitor on ice for 30 min. Cell lysates were centrifuged at 15,000 rpm at 4°C for 15 min, and postnuclear supernatant (PNS) was collected. The protein concentration was determined using a BCA protein assay. Protein was adjusted to 20 mg per sample and separated by 12% SDS–PAGE based on the molecular weight. The protein sample was transferred to a 0.45-µm polyvinylidine fluoride membrane and blocked with 5% nonfat milk in TBST buffer (150 mM NaCl, 0.1% Triton X-100, and 25 mM Trizma base, pH 7.6) for 1 h at room temperature. The membrane was incubated with primary antibody overnight at 4°C, rinsed six times for 5 min each, and probed with HRP-conjugated secondary antibody (anti-mouse, anti-rabbit, or anti-pig) for 1 h at room temperature. Most primary antibodies were diluted 1:1,000, and the secondary antibodies were diluted 1:5,000. The target protein signals were detected by enhanced chemiluminescence using an imaging system (ChemiScope 6100 Touch, ClinX). The acquired images were further processed with Adobe Photoshop CS6 and analyzed using ImageJ.
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