Phosphothreonine detection set

IP, WB

Contains a positive control for immunoblot applications as well as 25µg each of the following MAbs:
1E11 (Prod. No. ALX-804-168)
4D11 (Prod. No. ALX-804-169)
14B3 (Prod. No. ALX-804-170)

Western Blot (20µl/lane (mini gel) for HRPO/DAB detection, or 5µl/lane (mini gel) for HRPO/ECL detection. Do not use milk/casein for blocking and dilution! Replace by BSA! We recommend to use 1% BSA, 1% PVP-10 (polyvinylpyrrolidone)1% PEG 3500, 0.2% Tween 20 in 2x PBS)Suggested dilutions/conditions may not be available for all applications.Optimal conditions must be determined individually for each application.

Antibodies directed against phosphorylated epitopes recognize the phosphorylated amino acid in the context of the surrounding amino acid sequence. Recognition is therefore dependent on 2 criteria: 1) phosphorylation and 2) the surrounding amino acid motif. If one of the two criteria is not fulfilled, the antibody will not detect the phosphorylation site. Since the amino acid sequence varies between different phosphorylation sites, certain proteins – though phosphorylated – may not be detected by the antibody. Phosphorylation patterns in a given cell extract may differ when probed with different antibodies due to sequence specificity.

Positive Control Cell Lysate
Formulation: The pSer/pThr molecular weight marker contains rabbit muscle phosphoproteins isolated by Fe3+/IDA – affinity chromatography. Proteins are lyophilized from PBS/NaF/PEG/Sucrose and Bromophenolblue.
Reconstitution: Reconstitute by addition of 200 μl H2O. After complete solubilization add 200 μl 2x SDS-PAGE sample buffer, mix and incubate at 90ºC for 5 min. Aliquote and store frozen. Avoid repeated freeze/thaw cycles.
Application: The pSer/pThr molecular weight marker is recommended for immunoblot applications. Use 20μl molecular weight marker per lane. Note: Use BSA based blot incubation buffers. Milk, Casein and Blotto might interfere with antibody – antigen interaction.