Signals were detected by chemiluminescence (ECL). The membrane was washed in TTBS once for 15 minutes. The membrane was washed in TTBS twice for 5 minutes each. The membrane was incubated in the HRP-conjugated secondary antibody solution in TBS for 60 minutes at room temperature with gentle agitation. The membrane was immersed with the protein side up in the antibody solution in TBS and incubated overnight at 4☌ with gentle agitation.
The immunoblot membrane was blocked in 2.5% skim milk and 1.5% BSA solution in TTBS at room temperature for 60 minutes. Proteins were transferred onto nitrocellulose membrane by tank transfer and protein transfer was confirmed with Ponceau S staining.
#BETA ACTIN KDA WESTERN BLOT PLUS#
The Precision Plus Protein™ All Blue Prestained Standards from BioRad (161-0373) were used as molecular mass markers. 2.đ5 μg of boiled lysate were loaded and resolved on a 12% SDS-polyacrylamide gel.1.Ĝell/tissue total protein lysates were boiled in 1X SDS Sample Buffer containing 1% SDS and 1.25% β-mercaptoethanol at 95☌ for 5 minutes prior to loading.Synthetic peptide (aa 2-16 of mouse beta-actin) coupled to KLH via a C-terminally added cysteine. Rabbit serum albumin was added for stabilization. May cross-react to alpha- and gamma-actin due to sequence homology.Īffinity purified with the immunogen. Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC) Immunohistochemistry (Formalin-fixed Sections) (IHC (f)) Immunofluorescence (fixed cells) (IF/ICC) Immunohistochemistry (Frozen Sections) (IHC (fro)) Immunofluorescence (Cultured Cells) (IF (cc)) Immunohistochemistry (Paraffin-embedded Sections) (IHC (p))
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