Wes Manual for Quantification - This section summarizes how the system works, what to keep in mind when analyzing Wes' results and possible problems you might encounter
This section shows the titers of different antibodies for Wes. These titers are applicable to immunoprecipitations performed with 100 mg of MT302 powder and an elution performed in 60 ul of 2% SDS 40 mM Tris pH 8. In order to use these titles with other materials and antibodies, use MT302 as a reference. Here some of the pictures from the titriation process LINE-1 antibodies for Wes
Stock antibodies:
Mouse anti-ORF1 (4H1) - 2 µg/µl
Rabbit Anti-ORF2 9-7 - 0.35 µg/µl
Mouse Monoclonal M2 anti-Flag - 1 µg/µl
Material: 100 mg of MT302 powder, IPs using 10 µl of beads, elution in 60 µl
| IP | Antibody | Stock Dilution | Loaded Material | |||
| ORF1 beads | Anti-Flag | 1:100 | 5.8 mg of powder (3.5 µl of elution) | |||
| Anti-ORF2 | 1:10 | 5.8 mg of powder (3.5 µl of elution) | ||||
| Anti-ORF1(4H1) | 1:150 | 1.7 mg of powder (1 µl of elution) | ||||
| Anti-Flag beads | Anti-Flag | 1:100 | 0.83 mg of powder (0.5 µl of elution) | |||
| Anti-ORF2 | 1:100 | 1.7 mg of powder (1 µl of elution) | ||||
| Anti-ORF1(4H1) | 1:75 | 3.3 mg of powder (2 µl of elution) |
Material: 100 mg of PA-1 powder, IPs using 10 µl of beads, elution in 60 µl
| IP | Antibody | Stock Dilution | Loaded Material | |||
| ORF1 beads | Anti-ORF1(4H1) | 1:150 | 10 mg of powder (3 µl of elution) |
Antibody titration for extracts was performed using different samples (LD603, LD288, LD401 and MT302)
Different antibodies were tested:
Antibodies
Anti-ORF1 mouse (4H1) (2 mg/ml)
CDI 1A12 (0.5 mg/ml)
CDI 2B12 (0.5 mg/ml)
CDI 1B7 (0.5 mg/ml)
CDI 1A6 (0.5 mg/ml)
LaORF1 (0.5 mg/ml) and anti-His Tag MAB050H R&D
Anti-Flag Monoclonal M2 Mouse (3.5 mg/ml)
ORF2 9-7 (0.25 mg/ml) Glycerol
ORF2 9-7 (0.5 mg/ml) PBS
ORF2 9-7 (0.75 mg/ml) Glycerol
ORF2 11-1 (0.5 mg/ml) PBS
ORF2 11-1 (0.25 mg/ml) Glycerol
In this link you can find an excel summary document with the title for each antibody in each sample type: https://rockefeller.box.com/s/mwp85yoognhd0z8w8r7owslsaqhuovxr
The different runs are numbered in the slides, from this number they can be located in the excel document.
In this link you can download the slides that explain the content of each Wes run: https://rockefeller.box.com/s/spptzbd90ld0qxzvzisr8uxed8r98u5m
In this link you can find the Wes run files: https://rockefeller.box.com/s/89zeo6wgup4j11mxy9jdt379rntmyikz