Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase.

Immunoblotting is widely used in muscle physiology to determine protein regulation and abundance. However, research groups use different protocols, which may result in differential outcomes. Herein, we investigated the effect of various homogenization procedures on determination of protein abundance in human m. vastus lateralis biopsies. Furthermore, we investigated differences in abundance between young healthy males (n = 12) and type-2 diabetics (n = 4), and the effect of data normalization. Fractionated lysates had the lowest variation in total protein determination as compared to non-fractionated homogenates. Abundance of NKAα2, NKAß1, FXYD1, and glycogen synthase was higher (P < 0.05) in young healthy than in type-2 diabetics determined in both fractionated and non-fractionated samples for which normalization to the stain-free signal and/or standard curve did not affect outcomes. Precision and reliability of protein abundance determination between sample types showed a moderate to good reliability for these proteins, whereas the commonly used house-keeping protein, actin, showed poor reliability. In conclusion, fractionated and non-fractionated immunoblotting samples yield similar data for several sarcolemmal and cytosolic proteins, except for actin, which, therefore appears inappropriate for data normalization in immunoblotting of human skeletal muscle. Thus, fractionation does not seem to be a major source of bias when immunoblotting for NKA subunits and GS.

The effect of activated sludge treatment and catalytic ozonation on high concentration of ammonia nitrogen removal from landfill leachate.

This study adopted the combination of activated sludge treatment and catalytic ozonation technology to efficiently remove the high concentration of ammonia nitrogen from landfill leachate. Through optimizing the parameters continuously, the COD, NH4+-N, UV254 and colority respectively descended to 417.75 ± 6.72 mg/L, 9.77 mg/L, 1.98 ± 0.04 and 40 times, and 3D fluorescence also reduced significantly within 14 days. Target genes of AOB-amoA, nxrA, napA, nirS and nosZ analysis indicated that ammonia-oxidizing bacteria, nitrated bacteria, and denitrifying bacteria played a key role on nitrogen removal, aerobic denitrifying bacteria was dominated especially. The nitrogen removal process was as follows: catalytic ozonation converted nitrogen-containing organic matter into NH4+-N, then NH4+-N was converted into NO2--N and NO3--N with the action of ammonia oxidation, nitrification and catalytic ozonation. Finally, the denitrification microorganisms transformed NO3--N or NO2--N to N2. Therefore, this coupled process realized the nitrogen removal effectively from landfill leachate.