Cells were lysed using RIPA Buffer (Thermo Fisher Scientific, Waltham, MA) with a protease inhibitor cocktail kit (Sigma-Aldrich). Aliquots containing 30 μg of cell lysates were denatured in 5× Sample Buffer (Wako), electrophoretically resolved on SDS-polyacrylamide gels (Wako), and then transferred onto Immobilon polyvinyldifluoride membranes (Millipore, Billerica, MA). The membrane blots were blocked with 2% skimmed milk (Cell Signaling Technology, Danvers, MA) for an hour at room temperature, and then incubated with primary antibodies at 4 °C overnight. After the appropriate secondary antibodies were added for an hour, the signals were developed with Immun-Start AP Substrate (Bio-Rad) and observed by using LAS-3000 (Fujifilm, Tokyo, Japan).
Protease inhibitor cocktail kit
Manufactured by Merck Group
Sourced in United States
The Protease Inhibitor Cocktail Kit is a laboratory product designed to inhibit the activity of proteases, which are enzymes that break down proteins. The kit includes a ready-to-use solution of various protease inhibitors that can be added to protein samples to prevent unwanted proteolysis during extraction and purification procedures.
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Lab products found in correlation
3 protocols using protease inhibitor cocktail kit
1
Protein Extraction and Western Blotting
2
Molecular Signaling Pathways in Spinal Cord Tissue
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The rats were euthanized and the superficial dorsal horn (lumbar 4-6) tissues were dissected under an anatomical microscope. The tissues were then homogenized in ice-cold radioimmunoprecipitation assay buffer containing 25 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) with protease inhibitor cocktail kit (Sigma-Aldrich, St. Louis, MO). Then the tissues were centrifuged. The supernatant was then diluted to the same volume and applied to SDS-PAGE. Membranes were incubated with the rabbit anti-p-mTOR/p-S6K1/p-4EBP1antibodies; rabbit anti-mTOR /S6K1/4EBP1 antibodies; rabbit anti-p-PI3K p85/ anti- PI3K p85, respectively. All these primary antibodies were purchased from the Abcam Company (Cambridge, UK); and goat antirabbit secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Immunoreactive proteins were detected by enhanced chemiluminescence. The membrane was also processed to detect β-actin for equal loading. The optical density of protein bands was first analyzed using the NIH Scion image Software ImageJ (http://rsb. info.nih.gov/ij/ ), and values for densities of immunoreactive bands/β-actin band densities from the same lane were determined. Each of the values was then normalized to a control sample.
3
CsA Pathway and PCNA Expression
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CsA was purchased from Sandimmune ® , New Jersey, USA. A special kit for total RNA extraction was purchased from Gene All South Korea, Cat no 305-101. A universal cDNA synthesis kit for synthesizing the complementary DNA was prepared from Ampliqon Co, Denmark. The chemiluminescence detection kit was purchased from ECL; Thermo Scientific, Illinois, USA. The anti-proliferating cell nuclear antibody (PCNA) antibody was purchased from Dako Denmark A/S, Denmark. The protease inhibitor cocktail kit was prepared from Sigma-Aldrich S8820, USA.
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