Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for western blot analysis.
Western blotting of low molecular weight (Mr) acidic and basic isoelectric point (pI) proteins was studied to optimize detection sensitivities. Radioiodinated epidermal growth factor (EGF, Mr 6045, pI 4.4), transforming growth factor type alpha (TGF alpha, Mr 5623, pI 6.8), insulin-like growth factor (IGF-I, Mr 7649, pI 8.3), and basic fibroblast growth factor (bFGF, Mr 15,000-17,000, pI 9.6) all transferred with high efficiency (74.1 +/- 12.6%) to a positively charged nylon membrane. Sequential application of standard unoccupied site blocking, antibody incubation, and washing steps resulted in significant losses of all growth factors (46-98%). Basic FGF was retained best. Treatment of transfer membranes with 0.5% (v/v) glutaraldehyde prior to blocking and immunodetection increased the retention of the growth factors 1.5- to 12-fold over untreated controls. Without fixation, 100 ng of EGF, TGF alpha, and IGF-I were not detectable while 6.25-100 ng was identified on fixed membranes. The methods described were equally sensitive for detecting both acidic and basic pI proteins.
Karey
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Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Sirbasku
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D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral; D006133:Growth Substances / Q000302:isolation & purification*; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification*
D006133:Growth Substances; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D011506:Proteins; D005976:Glutaral