Skip to main content
Figure 2 | Molecular Medicine

Figure 2

From: Widespread Expression of Erythropoietin Receptor in Brain and Its Induction by Injury

Figure 2

EPOR IP using ctEPOR-AB and protein identification by mass spectrometry.

(A) Colloidal Coomassie staining and immunoblot of the same EPOR IP using ctEPOR-AB from UT-7 protein lysates. The overlay was used to determine the region to be excised from the Coomassie gel for subsequent mass spectrometric protein identification (area indicated by rectangles; abbreviations: FT = flow-through, IP = immunoprecipitation). (B) Amino acid sequence of EPOR (UniProtKB/Swiss-Prot P19235). Peptides identified by mass spectrometry are indicated in red. Note that large parts of the EPOR precursor sequence (indicated in italics) cannot be covered in a standard proteomic experiment with tryptic cleavage as they are either modified (amino acids 1–34, signal peptide; 57–89, N-glycosylation site), attached to the transmembrane domain (224–275) or too large (>5 kDa) to reveal useful information by mass spectrometric sequencing (379–453, 454–508). (C) Table with details on peptide identification. Columns show from left to right: numbering of tryptic peptides; numbering of amino acids according to the sequence in B; peptide sequence (c, carboxamidomethyl-Cys); observed and calculated mass of the singly protonated peptide; peptide mass deviation in ppm; PLGS score; number of b−y fragment ions; root mean square fragment mass deviation in ppm. (D) Immunoblot of EPOR IP using ctEPOR-AB from UT-7 lysates. In contrast to the IP used for mass spectrometry, EPOR was eluted from the beads in reducing conditions (Laemmli buffer with β-mercaptoethanol; abbreviations: FT = flow-through, IP = immunoprecipitation, Ig HC = immunoglobulin heavy chains, Ig LC = immunoglobulin light chains). The prominent band at around 40 kDa in both IP conditions has to be an immunoglobulin fragment eluted from the beads in the reducing condition only, since it was not eluted without β-mercaptoethanol (see subpanel 2A immunoblot).

Back to article page