Protein Expression Analysis Services: Wes and Western

Protein is where most drug targets sit and where most mechanisms act. Transcript measurement establishes what a cell is transcribing; protein measurement establishes what it has actually made, how much survives, and what state it is in. For programs whose mechanism involves phosphorylation, degradation, or stabilization, protein analysis is not a confirmatory endpoint but the primary one, since the transcript may not change at all.

Altogen Labs provides protein expression analysis by capillary electrophoresis based immunoassay using the Wes platform and by conventional Western blot, selecting the format according to sample availability and the required dynamic range. To discuss a protein endpoint, request a quote.

Choosing between capillary immunoassay and conventional blot

Capillary electrophoresis based immunoassay offers greater sensitivity and substantially lower sample consumption than conventional Western blot, with automated separation, immunodetection, and quantification producing digital output rather than a film image requiring densitometry. It matters most when available tissue is limited, as it frequently is with small tumors, scarce patient-derived material, or satellite cohorts where tissue is divided across several endpoints.

Conventional Western blot retains advantages where a broad molecular weight range must be visualized in a single run, where the target behaves unusually during capillary separation, or where an existing dataset was generated by blot and comparability matters more than sensitivity. Both are supported, and the choice is made per project rather than by institutional preference.

What is measured

Analysis covers total target protein abundance, post-translational modification state including phosphorylation, and the ratio between modified and total protein, which is usually the more interpretable measure since it separates a change in pathway activity from a change in protein level.

Antibody selection determines whether any of this is trustworthy. Antibodies are validated for the target, species, and matrix in use, since an antibody performing well in one cell type and application frequently does not transfer to tissue lysate from another species. Where a modification-specific antibody is used, specificity for the modified form is confirmed rather than assumed from the vendor description.

Applications

Applications include pharmacodynamic confirmation of target engagement, verification of knockdown in RNAi programs alongside transcript measurement, validation of overexpression in stable cell line development, pathway analysis in efficacy studies, and resistance characterization where pathway reactivation is the suspected mechanism.

Applied examples

Target engagement in tumor. Tumors from treated and vehicle animals are lysed and assessed for phosphorylation of a direct substrate of the drug target, with the phospho to total ratio establishing pathway inhibition independent of any change in total protein.

Knockdown at protein level. Following siRNA administration, target protein in the intended organ is quantified against vehicle controls and compared with transcript reduction in the same tissue, revealing whether protein turnover limits the functional effect of a transcript knockdown.

Resistance mechanism. A resistant line and its parental line are compared for target expression and for activation of candidate bypass pathways, distinguishing target-level from bypass-mediated resistance.

Quantification standards and reporting

Loading control selection, linear range determination, and replicate structure are defined before samples are run, since quantification outside the linear range of detection is not quantification. Where a comparison spans multiple runs, a bridging sample is included so that runs can be related to one another rather than compared as though generated together. Digital output and raw traces are retained alongside analyzed results. Antibody identity, dilution, and validation status are recorded in the report.

Send target, species, matrix, and available sample amount, or request a quote.