PhosphoThreonine Antibody Q7 (100 µg)

Protocol tips

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	1:500–1,000 dilution

Protocol tips
1:500–1,000 dilution

Publication protocol

Transfer the proteins from SDS-PAGE gel on to ProtranBA85, 0.45 μm nitrocellulose membrane (Whatman).
Examine the quality and efficiency of the transfer by staining the membrane with Ponceau S solution (Sigma) for 2 min (see Note 9).
Wash the membrane with dH2O.
Incubate the membrane in 5% milk-TBS-T for 15–20 min at room temperature.
Wash the membrane with 1× TBS-T for 5 min at room temperature.
Repeat (step 38) three times.
Incubate the membrane with 1:500–1,000 dilution of either phospho-serine (Q5) or phospho-threonine (Q7) antibodies, (Qiagen), in 2% BSA-TBS-T overnight at 4°C (see Note 10).
Wash the membrane three times with 1× TBS-T for 5 min at room temperature.
Incubate the membrane with 1:2,000 dilution of anti-secondary mouse antibody in 5% milk-TBS-T for 1 h (see Note 11) at room temperature.
Wash the membrane three times with 1× TBS-T for 5 min at room temperature.
Remove 1× TBS-T and then apply ECL plus (GE Healthcare) to nitrocellulose membrane for 2–3 min.
Drain nitrocellulose membrane of excess developing solution (do not let dry).
Wrap the blot in saran wrap.
Place the blot in the X-ray film cassette (see Note 12).
Expose the blots to X-ray films by placing X-ray film directly against the western blot for different lengths of time.

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Manufacturer protocol

Download the product protocol from Qiagen for PhosphoThreonine Antibody Q7 (100 µg) below.

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