A band was observed in the positive controls at the expected size (~50 kDa), which is lower in the negative controls.
Validierungsbilder
Figure 1: Western Blot for Tubulin, Beta, 3 (TUBB3). Grey arrowhead indicates the expected molecular weight of ~50 kDa.
Protokoll
Primärantikörper
Antigen: Tubulin, Beta, 3 (TUBB3) (AA 443-450)
Catalog number: ABIN1742553
Lot number: 302302/3
Dilution: 1:1000
Sekundärantikörper
Antibody: Donkey anti-Rabbit IgG Antibody (HRP)
Lot number: F0613
Dilution: 1:10,000
Full Protocol
1. Cell/tissue total protein lysates were boiled in 1X SDS Sample Buffer containing 1% SDS and 1.25% β-mercaptoethanol at 95°C for 5 minutes prior to loading.
2. 15 μg of boiled lysate were loaded and resolved on a 12% SDS-polyacrylamide gel.
3. The Precision Plus Protein™ All Blue Prestained Standards from BioRad (161-0373) were used as molecular mass markers.
4. Proteins were transferred onto nitrocellulose membrane by tank transfer and protein transfer was confirmed with Ponceau S staining.
5. The immunoblot membrane was blocked in 2.5% skim milk and 1.5% BSA solution in TTBS at room temperature for 60 minutes.
6. The membrane was washed in TTBS twice for 5 minutes each.
7. The membrane was immersed with the protein side up in the antibody solution in TBS and incubated overnight at 4°C with gentle agitation.
8. The membrane was rinsed twice with TTBS.
9. The membrane was washed in TTBS twice for 5 minutes each.
10. The membrane was washed in TTBS once for 15 minutes.
11. The membrane was incubated in the HRP-conjugated secondary antibody solution in TBS for 60 minutes at room temperature with gentle agitation.
12. The membrane was rinsed twice with TTBS.
13. The membrane was washed in TTBS twice for 5 minutes each.
14. The membrane was washed in TTBS once for 15 minutes.
15. Signals were detected by chemiluminescence (ECL). The blot was scanned for 320 seconds.
16. The membrane was rinsed three times with TTBS.
17. Repeated Steps 4-15 with the loading control antibody and its matching secondary antibody. The blot was scanned for 160 seconds.
Anmerkungen
- No experimental challenges noted.
Format
Liquid
Buffer
PBS
Handhabung
Crude antisera are more robust than monoclonals. With anti-microbials added, they may be stored at 4 °C. Serum does not contain active proteases, in fact, serum itself contains a powerful cocktail of protease inhibitors. Frozen storage (-20 °C),however, is preferable.
Lagerung
4 °C/-20 °C
Informationen zur Lagerung
Unlabeled antibodies are stable in this form without loss of quality at ambient temperatures for several weeks or even months. They can be stored at 4 °C for several years.
Kaczor, Rakus, Mozrzymas: "Neuron-astrocyte interaction enhance GABAergic synaptic transmission in a manner dependent on key metabolic enzymes." in: Frontiers in cellular neuroscience, Vol. 9, pp. 120, (2015) (PubMed).