Lars10th January 2020 at 11:12am
AIM/HYPOTHESIS
To see whether newly conjugated beads efficiently pull out ORF1p. Comparison will be made with an old batch of anti-ORF1p beads and two different cell lines.
MATERIALS
| Name | Company | Cat. nr. | Date received/made | Comments | ||||
|---|---|---|---|---|---|---|---|---|
| Old batch a-ORF1p beads | Oct 22 2019 | |||||||
| New batch a-ORF1p beads | Dec 3 2019 | See Conjugation of new anti-ORF1p antibodies and Dynabeads | ||||||
| MT302 Puro cell powder | Sep 09 2013 | |||||||
| MT302 Light cell powder | May 02 2014 | |||||||
| a-ORF1p abs | Abmart | ab solution made Oct 31 2019 | mouse, 1:5000 | |||||
| HRP anti mouse ab | 1:10000 | |||||||
| Sypro stain | +syprofix and syprowash |
- Filtered extraction buffer (20mM HEPES, pH7.4, 1% TritonX-100, 500mM NaCl, 1x protease inhibitor)
- Scale: 50mg cell powder per IP reaction with 5ul of beads
METHODS
IP was performed according to Immunoprecipitation with antibody conjugated Dynabeads from cell powder. Both cell powders were combined with both batches of beads.
Samples were run on 4-12% Bis-Tris gel and analyzed using a Sypro staining and anti-ORF1p antibodies. [protocols will follow]
RESULTS
Gel
Western blot
DISCUSSION