After HeLa cells were treated with 10 uM dexamethasone for 1 h before stimulation with 100 uM histamine, whole cell extracts were prepared at various time points as described previously (9) . Ten µg of total protein per sample was separated on a 10% SDS-PAGE gel then transferred to a nitrocellulose membrane (Bio-Rad). The membrane was briefly rinsed in Tris-buffered saline with 0.1% Tween 20 (TBS-T) then incubated for 1 h at room temperature in TBS-T with 3% BSA (Sigma). The membrane was then incubated overnight at 4℃ with one of the following primary antibodies : PKCδ (C-20), sc-937, 1:1000 ; ERK (K-23), sc-94, 1:1000 ; phospho-ERK (E-4), sc-7383, 1:1000, (Santa Cruz Biotechnology) ; phospho-PKCδ (Tyr311), #2055S, 1:500 (Cell Signaling). The membrane was then incubated in the appropriate secondary antibody (goat anti-rabbit IgG (H ± L)-HRP conjugate (#170-6515, 1:10,000, Bio-Rad) or Immun-Star goat anti-mouse-HRP conjugate (#170-5047, 1:10,000, Bio-Rad) and proteins visualized using Immobilon Western Chemiluminescent HRP substrate (Millipore).
Immun star goat anti mouse hrp conjugate
Manufactured by Bio-Rad
The Immun-Star goat anti-mouse-HRP conjugate is a laboratory reagent used in immunoassays. It contains horseradish peroxidase (HRP) conjugated to goat-derived antibodies that are specific to mouse immunoglobulins. This product can be utilized to detect and quantify mouse-derived proteins or antigens in various experimental applications.
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2 protocols using immun star goat anti mouse hrp conjugate
1
Dexamethasone-Induced Histamine Response
2
Western Blot Protein Quantification
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Twenty to thirty micrograms of WCL or 5–10μl immunoprecipitated proteins were loaded on a precast 4–12% NuPAGE Tris-glycine gel, run for 2–3 h at 100–130 volts. The gel was transferred and the membrane was blotted with primary antibodies, as indicated. The information for the primary antibodies used herein is listed in Supplementary Table S4 . After washing, Immun-Star Goat anti-mouse-HRP conjugate (Bio-Rad) or Immun-Star Goat anti-rabbit-HRP conjugate (Bio-Rad) secondary antibody was used. The membrane was illuminated and the bands were quantified using Image Lab (Bio-Rad).
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