Considerations when quantitating protein abundance by immunoblot

Abstract

The development of the immunoblot to detect and characterize a protein with an antisera, even in a crude mixture, was a breakthrough with wide-ranging and unpredictable applications across physiology and medicine. Initially, this technique was viewed as a tool for qualitative, not quantitative, analyses of proteins because of the high number of variables between sample preparation and detection with antibodies. Nonetheless, as the immunoblot method was streamlined and improved, investigators pushed it to quantitate protein abundance in unpurified samples as a function of treatment, genotype, or pathology. This short review, geared at investigators, reviewers, and critical readers, presents a set of issues that are of critical importance for quantitative analysis of protein abundance: 1) Consider whether tissue samples are of equivalent integrity and assess how handling between collection and assay influences the apparent relative abundance. 2) Establish the specificity of the antiserum for the protein of interest by providing clear images, molecular weight markers, positive and negative controls, and vendor details. 3) Provide convincing evidence for linearity of the detection system by assessing signal density as a function of sample loaded. 4) Recognize that loading control proteins are rarely in the same linear range of detection as the protein of interest; consider protein staining of the gel or blot. In summary, with careful attention to sample integrity, antibody specificity, linearity of the detection system, and acceptable loading controls, investigators can implement quantitative immunoblots to convincingly assess protein abundance in their samples.

Keywords: Western blot; antibody-antigen; immunoblot; immunodetection; quantitation.

Fig. 1.

Consider sample integrity. A: illustration showing that the percentage of normal cardiac myocytes can be significantly decreased in infarcted myocardium versus normal myocardium. B: illustration of the effect of freezing (versus not freezing) mouse kidneys before homogenization on the detection of the Na⁺/H⁺ exchanger isoform 3 (NHE3) epitope, a measure of the apparent abundance of NHE3. C: illustration of the near total loss of renal transporter epitope recognition when rat kidneys are frozen before preparation of homogenates. See text for details and discussion. NKCC, Na⁺-K⁺-2 Cl⁻ cotransporter; NCC, Na⁺-Cl⁻ cotransporter.

Fig. 2.

Consider the specificity of the antibody for the target. SPAK [sterile 20 (STE20)/SPS1-related proline/alanine-rich kinase] was detected first with an anti-NH₂-terminal epitope antiserum, which detects full-length SPAK (FL-SPAK), then a red-tagged secondary; the blot was reprobed with an anti-COOH-terminal epitope antisera that detects FL-SPAK, smaller SPAK 2, and an even smaller kidney-specific SPAK isoforms (KS-SPAK), then a green-tagged secondary. Bottom: illustration of the image obtained when both signals were detected at the same time; yellow indicates detection at the same location. SPAK is expressed in testes as a single full-length isoform (FL-SPAK) included as a positive control, is absent in a SPAK knockout mouse (SPAK-KO), included as a negative control, and all three isoforms are present in wild-type mouse kidney. Nonspecific background bands (white arrows) were identified based on expression in the SPAK-KO. See text for details and discussion.

Fig. 3.

Consider the linearity of the detection system. A: illustration of the immunodetection of sodium pump catalytic isoform subunits (α₁, α₂, α₃) and the Na⁺/Ca²⁺ exchanger (NCE) from human heart detected between 20 and 100 μg/lane. [Redrawn from Wang et al. (27).] B: illustration of the immunodetection of sodium pump (NKA) α₁-subunit between 0.12 and 0.5 μg/lane and β₁-subunit between 3 and 48 μg/lane in rat kidney homogenates from cortex and medulla. See text for details and discussion.

Fig. 4.

Consider the loading controls: part 1. A: illustration of a single immunoblot constructed from two SDS-PAGE gels loaded with 6 μg/lane (top of blot) and 3 μg/lane (bottom part of blot) kidney homogenate and probed with antiserum to claudin 10 and a secondary antiserum tagged red. The quantitation, indicated to the right of the blot, demonstrates linearity of the detection system: Specifically, signal intensity increases 1.7-fold with sample doubling. B: image of the same blot reprobed with an anti-actin (pan-actin) antiserum and a secondary antiserum tagged green. The actin signal intensity does not change with sample doubling. We conclude that actin is not an acceptable loading control for this application; rather it is “overloaded” at 3 and 6 μg kidney homogenate/lane. See text for further details and discussion.

Fig. 5.

Consider the loading controls: part 2. Direct protein staining of the gel or blot is an alternative to the loading control to assess whether the same amount of protein is loaded per lane. Top: image of a stained post SDS-PAGE gel (not blot) of renal homogenate samples from eight rat kidneys loaded at 7.5 μg/lane. Arrows indicate the 5 unidentified protein bands arbitrarily chosen for quantitation to determine whether the 8 samples were equivalently loaded. Values were normalized to the mean value of each band in the 8 samples defined as 1.0. Bottom: illustration of the relative density of each of the 5 arbitrary bands (color coded) in each sample as well as the average of the 5 bands (black). The results indicate that samples 1, 2, 3, 6, and 8 are very close to equivalent with average relative density of 1.0 while 4, 5, and 7 deviate, by <13%. See text for further details and discussion.