![]() ![]() Hence, storage at 4 ☌ for prolonged periods (i.e. Then aliquot and store at -20 ☌ until use.Īffinity purified antibodies are less robust than antisera, since protease inhibitors are also removed during purification. In our experience, we find that the level of actin does vary widely between different cell and tissue types.įor reconstitution add 100 µL H2O to get a 1mg/ml solution of antibody in PBS. The β-actins and γ-actins co-exist in most cell types as components of the cytoskeleton, whereas the α-actin are found in muscle tissues. Three main groups of actin have been identified. Lower molecular weight bands of approximately 28-30 kDa were also observed in the mouse kidney and spleen tissue samples. The blot was scanned for 160 seconds.Įxperimental Notes A protein band was observed at the expected target size of β−Actin of 42 kDa in all mouse extracts tested. Repeated Steps 4-14 with the loading control antibody and its matching secondary antibody. Repeated Steps 4-14 with the loading control antibody (for β3−Tubulin). The membrane was washed in TTBS 3 times for 5 minutes each. Incubated in Acidic Glycine Stripping Buffer at room temperature with gentle agitation for 3 times, 10 minutes each. The membrane was rinsed three times with TTBS. 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. 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.Custom Recombinant Antibody (rAbs) Services. ![]() Annexin V-FITC Apoptosis Detection Kits. ![]()
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