Q: Why am I getting high background?
A: The high background is likely due to having too much free DNPH remaining on the membrane. DNPH is a sticky molecule and free DNPH can stick to the membrane and be detected by the antibody, causing high background. To avoid background, we recommend incubating the membrane with DNPH for only 5 minutes followed by extensive washing of the membrane with 2N HCl at least three times. The number of washes can be increased from three to five if necessary.
Q: Why does the membrane require washing with methanol and HCl?
A: After the DNPH reaction with the carbonyl groups, there is still an excess of free DNPH remaining. DNPH is a very sticky molecule and requires extensive washing with methanol and HCl to remove free DNPH from the membrane. The antibody binds to free DNPH in addition to protein carbonyl-DNP complexes and will result in high background if the free DNPH is not completely washed away.
Q: What is the best way to prepare protein lysates?
A: The lysate preparation will not affect the assay because the DNPH reaction and antibody detection steps occur after the SDS-PAGE and protein transfer. The lysates can be made in RIPA buffer or any other preferred lysis buffer.
Q: Do you have any recommendations for the electrophoresis and transfer steps?
A: The SDS-PAGE and transfer steps are the same as any standard Western blot and any general lab protocol can be used. After the protein transfer, the protocol in the manual should be followed.
Q: Can the membrane be stored overnight before performing the DNPH reaction?
A: The PVDF membrane can be stored overnight at 4ºC before the DNPH reaction. If stored after the DNPH reaction it will be difficult to wash away the free DNPH and will result in high background.
Q: What is the stability of carbonylated proteins?
A: Protein carbonyl is a very stable modification and protein samples can be stored for at least 6 months at -80ºC before degradation starts to occur.