COMPARISON OF HOUSEKEEPING PROTEIN AND TOTAL PROTEIN NORMALIZATION IN WESTERN BLOTTING ANALYSIS OF ENDOTHELIAL CELL LYSATE AND VASCULAR HOMOGENATE

Highlights

• Acid Black 1 and Fast Green FCF are the most sensitive dyes for total protein staining in endothelial cell lysate or vascular homogenate, detecting as low as ≥ 1 µg protein.
• Staining with antibodies to β-actin, TBP, and PCNA has the highest sensitivity in endothelial cell lysate, detecting as low as ≥ 1–2 µg protein, ≥ 2–4 µg protein, and ≥ 4 µg protein, respectively.
• Total protein staining can be employed for the signal normalization in Western blotting of endothelial cell lysate or vascular homogenate if coefficient of variation is ≤ 20% (in case of endothelial cell lysate) or ≤ 25% (in case of vascular homogenate).

Aim. To compare the sensitivity of housekeeping proteins and total protein staining for the signal normalisation during Western blotting of endothelial cell (EC) lysate and vascular homogenate.

Methods. Serial dilutions of EC lysate or vascular homogenate (1, 2, 4, 8, 16, and 32 µg protein) were loaded into bis-tris polyacrylamide gel and stained for EC housekeeping proteins: platelet endothelial cell adhesion molecule 1 (CD31/PECAM1), β-actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), TATA-binding protein (TBP), proliferating cell nuclear antigen (PCNA), histone H3, and cytochrome c oxidase subunit 4 (COX4). Housekeeping protein expression was detected using secondary antibodies conjugated with 680RD and 800CW fluorophores or horseradish peroxidase. Total protein staining was performed using Acid Black 1, Fast Green FCF, Ponceau S, Congo Red, nigrosine, Revert 700, and 2,2,2-trichloroethanol.

Results. Acid Black 1 and Fast Green FCF were the most sensitive (≥ 1 µg protein) dyes for total protein staining in EC lysate or vascular homogenate on polyvinylidene fluoride or nitrocellulose membranes. Among the housekeeping proteins of EC lysate and vascular homogenate, the highest sensitivity was detected for β-actin (≥ 1–2 µg protein), TBP (≥ 2–4 µg protein), and PCNA (≥ 4 µg protein). Total protein staining was more sensitive than β-actin or TBP staining. Total protein staining of EC protein lysate showed maximum and median coefficient of variation of ≈ 20% and ≈ 12% for the EC lysate and ≈ 40% and ≈ 17% for vascular homogenate.

Conclusion. The most sensitive dye for total protein staining in EC lysate and vascular homogenate is Acid Black 1, the most sensitive cytoplasmic housekeeping protein is β-actin, and the most sensitive nuclear housekeeping protein is TATA-binding protein (TBP). For the respective normalization, total protein staining can be employed if coefficient of variation is ≤ 20% (for EC lysate) or ≤ 25% (for vascular homogenate).

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