Western blot assays [36 (link)] were carried out using minigels [37 ] transferred to PVDF membranes (Immun-Blot®, BioRad Laboratories Inc.) using a Trans-Blot® Turbo™ Transfer System (BioRad Laboratories Inc.). Transfer conditions were 25 V during 30 minutes. Membranes were washed in Tris-buffered saline with 1% Tween 20 (TBST) and blocked with 5% skimmed milk (Sigma-Aldrich Co.) in TBST overnight. After blocking, membranes were washed 3 times with TBST and incubated for 2 hours with M22.8 hybridoma supernatant (1:10 dilution in TBST with 2,5% skimmed milk) at room temperature (RT). For colorimetric Western blotting assays, goat anti-mouse IgG antibodies conjugated to AP (Dako) diluted 1:1,000 in TBST with 2.5% skimmed milk were used as secondary antibodies (1.5 hours, RT). Goat anti-mouse IgG antibodies conjugated to horse rabbit peroxidase (HRP) (Dako) diluted 1:50,000 in TBST with 2.5% skimmed milk were used as secondary antibodies (1 hour, RT) for chemiluminescent Western blot assays. Colorimetric Western blot assays were revealed with 1-Step NBT/BCIP (Thermo Scientific) whereas the Immun-Star™ WesternC™ Chemiluminescent Kit (BioRad Laboratories Inc.) was selected for chemiluminescent assays. Protein bands were analyzed using the ChemiDoc XRS imaging system in conjunction with ImageLab software (BioRad Laboratories Inc.).
Skimmed milk
Manufactured by Agilent Technologies
Sourced in Denmark
Description not available
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
1 step nbt bcip, by Thermo Fisher Scientific (1 mentions)
Immun star westernc chemiluminescent kit, by Bio-Rad (1 mentions)
Immun blot, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Agilent Technologies (1 mentions)
Donkey serum, by Agilent Technologies (1 mentions)
2 protocols using skimmed milk
1
Western Blot Assay Protocol
2
Immunohistochemical Analysis of GLP-1R
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Paraffin sections of kidney and pancreas tissue from normotensive WKY rats and prehypertensive SHRs were made. Sections were deparaffinated and rehydrated in double-distilled water. Sections were treated with 0.1% pronase in PBS at 37°C for 10 min and rinsed in Tris-buffered saline (TBS). Thereafter, sections were treated with 1% H 2O2 in TBS for 15 min, washed in TBS with 0.05% Tween (TBS-T), blocked with avidin for 10 min (Dako, Glostrup, Denmark), washed with TBS-T, blocked with biotin for 10 min (Dako), washed with TBS-T, and preincubated with 3.2 mg/mL poly-L-lysine, 3% BSA, 7% donkey serum, and 3% skimmed milk (Dako) for 30 min. Sections were incubated overnight with primary biotinylated mouse GLP-1R antibody, which was a generous gift from Charles Pyke (7F382A, Novo Nordisk, Måløv, Denmark). Thereafter, sections were washed three times for 10 min each in TBS-T followed by treatment with Vectastain ABComplexHRP in TBS for 30 min and washed again three times for 10 min each. Sections were developed with DAB ϩ (Dako), counterstained with hematoxylin, rinsed in water, dehydrated, and mounted.
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