DRG and lumbar spinal cord of 9-week-old WT and Grp cKO mice were dissected on ice and quickly frozen in −80 °C. Soluble cell plasma proteins were extracted as described75 (link). Samples were removed into a microtube containing ice-cold sample buffer (20 mM Tris-HCl [pH 7.4], 1 mM dithiothreitol, 10 mM NaF, 2 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 5 mM microcystin-LR, and 0.5 mM phenylmethylsulfonyl fluoride), and homogenized by sonication. Homogenates were centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was used for analysis. Protein concentration was determined using BCA assay (Thermo Scientific). For each sample, 10 µg of total protein were separated on SDS NuPAGE Bis-Tris 4–12% gels (Life Technology) in MES running buffer (Life Technology) and transferred to polyvinylidene fluoride membrane (Life Technology). The blots were blocked in blocking buffer (5% nonfat dry milk in PBS and 0.1% Tween 20) for 1 h at room temperature and incubated with rabbit anti GRP (ImmunoStar, 1:5,000), for 16 h at 4 °C. This was followed by 1 h incubation in goat horseradish peroxidase-linked secondary antibodies (Santa Cruz) at 1:2500. Immunoblots were developed with the enhanced chemiluminescence reagents (Amersham).
Rabbit anti grp
Manufactured by Immunostar
Sourced in United States
Rabbit anti-GRP is a polyclonal antibody raised in rabbits against the Gastrin-Releasing Peptide (GRP) protein. This antibody is designed for use in various immunological techniques, such as Western blotting, immunohistochemistry, and ELISA, to detect and quantify GRP in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (2 mentions)
Mes running buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (2 mentions)
Sds nupage bis tris 4 12 gels, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Rabbit anti actin, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Abcam (1 mentions)
Rabbit anti avp, by Bachem (1 mentions)
Rabbit anti vip, by Immunostar (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
3 protocols using rabbit anti grp
1
Protein Extraction and Western Blot Analysis of GRP in Mouse Tissues
2
Protein Extraction and Western Blot Analysis
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Cervical and thoracic DRGs were dissected on ice and quickly frozen in −80℃. Samples were removed into a microcentrifuge tube containing ice-cold sample buffer (20 mM Tris-HCl [pH 7.4], 1 mM dithiothreitol, 10 mM NaF, 2 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 5 mM microcystin-LR, and 0.5 mM phenylmethylsulfonyl fluoride), and homogenized by sonication. Homogenates were centrifuged at 12,000 × g for 30 min at 4℃. The supernatant was used for analysis. Protein concentration was determined using BCA assay (Thermo Scientific). For each sample, 10 µg of total protein were separated on SDS NuPAGE Bis-Tris 4–12% gels (Life Technology) in MES running buffer (Life Technology) and transferred to polyvinylidene fluoride membrane (Life Technology). The blots were blocked in 5% bovine serum albumin in PBS and 0.1% Tween 20 for 1 h at room temperature and incubated with rabbit anti-GRP (ImmunoStar, 1:5000), or rabbit anti-Actin (Sigma, 1:50,000) for 16 h at 4℃. This was followed by 1 h incubation in donkey horseradish peroxidase-linked secondary antibodies (Santa Cruz, 1:2500). Immunoblots were developed with the enhanced chemiluminescence reagents (Thermo Scientific). Band intensities were measured using Kodak 1D (version 3.6) and Actin served as internal control for normalization.
3
Immunostaining for SCN and Cartilage Clock Proteins
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For the SCN, mice were sacrificed at ZT 9 (lights off for animals is ZT12), and brains post-fixed in 4% PFA (in 0.01M phosphate buffer). Immunostaining protocol carried out as previously described [12 (link)]. Primary antisera: rabbit anti-AVP (1:1000; Bachem, USA), rabbit anti-VIP (1:1000, Immunostar, USA), rabbit anti-GRP (1:1000; Immunostar, USA), rabbit anti-BMAL1 (1:500; generated in-house) [43 (link)]. Secondary antiserum: Alexa 647(1:500, Life Technologies, USA). Sections mounted with Vectashield containing DAPI (Vector Labs, USA). For cartilage, immunostaining protocol carried out as previously described [14 (link)]. Primary antiserum: Anti-BMAL1 [43 (link)]. Secondary antiserum, Alexa Fluor 647 (Abcam, UK). Sections mounted with Vectashield containing DAPI (Vector Labs, USA).
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