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. Author manuscript; available in PMC: 2024 Oct 1.
Published in final edited form as: J Immunol Methods. 2023 Aug 18;521:113540. doi: 10.1016/j.jim.2023.113540

Figure 2. Commercially acquired phospho-specific Abs tested using Vanilla MILKSHAKE.

Figure 2.

Blots were incubated with primary Abs from seven vendors diluted to vendor recommended concentrations for four different desired target sequences A) phospho-AKT (Ser473) B) phospho-Stat3 (Tyr705) C) phospho-ERK1 (Thr202/Tyr204) D) phospho-JNK1 (Thr183/Tyr185). Each IgG Ab binds the phosphorylated residues listed for each target which are present in the phospho conjugated modMBP (A and B) or the dual phosphorylated MILKSHAKE in Lane 2P or the single-phosphorylated MILKSHAKE proteins (in C and D). In some cases, Abs also bind non-phospho conjugated modMBP (see yellow arrows in A and Lane 0P in D (qualitative data). Each lane in A and B was loaded with 3 μg MILKSHAKE protein only, and each lane in C and D was loaded with 10 μg MILKSHAKE protein only. Exposure times are listed on each blot. Lane M: Precision Plus Protein Standard [BioRad] Lane MBP: unconjugated modMBP, negative control, Lane 2P: dual phosphorylated, Lane 0P: dual non-phosphorylated