RIPA lysate (R0278; Sigma-Aldrich; Merck KGaA) and a protease inhibitor were used to lyse cells (1×106) and extract total protein (S8830; Sigma-Aldrich; Merck KGaA). Each sample's total protein (50 µg per lane) was separated on a 10% SDS-PAGE at 120 V for around 1.5 hours. The protein suspension was mixed with 1X antibody solution, which was then transferred to a PVDF membrane and sealed with 5% skim milk for 1 hour at room temperature. The cell membranes were sealed with skim milk and then treated with a primary antibody overnight at 4°C (Sigma-Aldrich; Merck KGaA). The membranes were incubated with the matching secondary antibodies for 1 hour at room temperature the next day, after being washed with Tris-buffered saline and Tween 20 (TBST; Sigma-Aldrich, St. Louis, MO, USA). To find the proteins, Cell Signaling Technology, Inc.'s SignalFire™ ECL reagent (Catalog #6883) was used. Protein blot results were optical density analyzed and quantified using ImageLabTM software (version 3.0) from Bio-Rad Laboratories Inc.
Tris buffered saline and tween 20 tbst
Manufactured by Merck Group
Sourced in United States
Tris-buffered saline and Tween 20 (TBST) is a buffer solution commonly used in various laboratory techniques. It is a mixture of Tris-base, sodium chloride, and the non-ionic detergent Tween 20. TBST serves as a general-purpose buffer and is often used for washing and blocking steps in immunoassays, protein blotting, and other biochemical procedures.
Automatically generated - may contain errors
Lab products found in correlation
S8830, by Merck Group (1 mentions)
Image lab software version 3, by Bio-Rad (1 mentions)
Ripa lysate, by Merck Group (1 mentions)
Sodium citrate buffer, by Merck Group (1 mentions)
Mouse anti insulin, by Abcam (1 mentions)
Goat serum, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rabbit anti glucagon, by Abcam (1 mentions)
2 protocols using tris buffered saline and tween 20 tbst
1
Western Blot Protein Analysis Protocol
2
Quantitative Immunofluorescence Analysis of Islet Composition
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After formalin fixation and paraffin embedding (FFPE), the blocks obtained were sectioned to obtain 5 µm slices which were then mounted on slides and stained using standard protocols (19) .
Briefly, the slides were deparaffinized and rehydrated using xylene and a serial dilution of alcohols. They were then heated in a 10mM solution of sodium citrate buffer (Sigma Aldrich, St. Louis, MO), rinsed in a mixture of Tris-Buffered Saline and Tween 20 (TBST) (Sigma Aldrich, St. Louis, MO) and blocked with 10% goat serum (Sigma Aldrich, St. Louis, MO). Following this, primary antibodies against porcine insulin (Mouse anti-insulin, Abcam, Cambridge, MA) and glucagon (Rabbit anti glucagon, Abcam, Cambridge, MA) were added. Appropriate secondary antibodies against insulin (Anti Ms IgG-Alexa 488(green), Abcam, Cambridge, MA), and glucagon (Anti Rb IgG-Alexa 555(red), Abcam, Cambridge, MA) were added. Afterwards, the tissue was mounted with mounting media containing DAPI (4', 6-diamidino-2-phenylindole) (Vectashield, Vector Laboratories, Burlingame, CA) which binds to A-T rich regions in the DNA resulting in blue fluorescence under a fluorescence microscope. A minimum of 100 islets were imaged and analyzed using image analysis algorithms (ImageJ) to determine the percentage of insulin positive and glucagon positive cells within islets in a given sample.
Briefly, the slides were deparaffinized and rehydrated using xylene and a serial dilution of alcohols. They were then heated in a 10mM solution of sodium citrate buffer (Sigma Aldrich, St. Louis, MO), rinsed in a mixture of Tris-Buffered Saline and Tween 20 (TBST) (Sigma Aldrich, St. Louis, MO) and blocked with 10% goat serum (Sigma Aldrich, St. Louis, MO). Following this, primary antibodies against porcine insulin (Mouse anti-insulin, Abcam, Cambridge, MA) and glucagon (Rabbit anti glucagon, Abcam, Cambridge, MA) were added. Appropriate secondary antibodies against insulin (Anti Ms IgG-Alexa 488(green), Abcam, Cambridge, MA), and glucagon (Anti Rb IgG-Alexa 555(red), Abcam, Cambridge, MA) were added. Afterwards, the tissue was mounted with mounting media containing DAPI (4', 6-diamidino-2-phenylindole) (Vectashield, Vector Laboratories, Burlingame, CA) which binds to A-T rich regions in the DNA resulting in blue fluorescence under a fluorescence microscope. A minimum of 100 islets were imaged and analyzed using image analysis algorithms (ImageJ) to determine the percentage of insulin positive and glucagon positive cells within islets in a given sample.
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