Phytoene desaturase (PDS) is commonly used as a reporter gene for RNA silencing experiments in plants. I would be very pleased to receiving from people that produced/bought an anti-PDS antibody a small amount for some western blot assays. Tomato and Nicotiana benthamiana are plant hosts I am working with. Thank you for your support.
Monoclonal ANTI-FLAG M2 is a mouse derived, affinity purified IgG1 monoclonal antibody that binds to fusion proteins containing a FLAGÒ peptide sequence.
The M2 antibody will recognize a FLAG peptide sequence at the N-terminus, Met-N-terminus, C-terminus, or internal sites of a fusion protein. Binding of the M2
antibody is not calcium dependent. Monoclonal ANTI-FLAG M2 is useful for detection,
identification, and capture of fusion proteins containing a FLAG peptide sequence by common immunological procedures, such as Western blotting, immunofluorescence, and immunoprecipitation.
Use the following method
Separate fusion proteins containing a FLAG peptide sequence from sample lysates using a standard SDS-PAGE protocol. Load 2.5–10 mg of total protein lysate per lane.
2. Transfer proteins from the gel to a nitrocellulose membrane, ImmobilonÒ
-P, or other polyvinylidene difluoride (PVDF) membrane. The PVDF membrane may provide greater downstream sensitivity.
3. Wash the blot in at least 0.5 mL/cm2 of purified water for 2–3 minutes employing gentle agitation (50–60 rpm).
4. Block the blot with at least 0.5 mL/cm2 of Tris Buffered Saline (TBS), pH 8.0, with 3% nonfat milk for 30 minutes at room temperature, employing gentle agitation.
5. Remove the blocking agent and wash once with 0.5 mL/cm2
of Tris buffered saline, pH 8.0.
Add the desired concentration of monoclonal antibody to the blot. A final antibody concentration of 1 mg/mL (1:1,000 dilution of the antibody as supplied) in at least 0.5 ml/cm2 of TBS with 3% nonfat milk is suggested. Incubate at room temperature for 30 minutes employing gentle agitation.
7. Decant off the Monoclonal ANTI-FLAG M2 solution and wash once with at least 0.5 ml/cm2 of TBS, pH 8.0.
8. Add the secondary antibody in the form of AntiMouse IgG-Peroxidase, Catalog Number A9044 or equivalent. The concentration of secondary antibody must be optimized based on the substrate being used. For detection using hemiluminescent
Peroxidase Substra-1, a final secondary antibody dilution of 1:30,000 should be employed. Specifically, it is suggested the antibody as supplied be diluted in at least 0.5 mL/cm2 of TBS with 3% nonfat milk. Incubate the blot employing
gentle agitation at room temperature for 30 minutes.
9. Wash the blot at least three times for a total of 15 minutes (5 minutes per wash) in TBS with 0.05% TWEEN® 20, pH 8.0, Catalog Number T9039. Agitate gently, employing at least 0.5 ml/cm2 of wash solution. 10. Develop the blot using ChemiluminescentPeroxidase Substrate-1 or an equivalent reagent for 5 minutes. Do not agitate the blot during this incubation step. Drain briefly and wrap in plastic
film.
.
Indirect Immunofluorescent Cytochemical Staining Monoclonal ANTI-FLAG M2 may be utilized in immunocytochemical staining procedures when used in conjunction with a labeled secondary antibody (indirect).6 A generic procedure for adherent cell
staining is described, using immunofluorescence, employing an Anti-Mouse IgG-FITC conjugate as the label
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