Lane 3, nanoparticles loaded without boiling show faint protein band bound to the AuNPs at 116 kDa mark.
However, in the sample that was not boiled (lane 3), a faint protein band bound to AuNP was seen at the 116 kDa level.
In the latter case, bands uniquely bound to GST-ErbB4 were further analyzed by Mass Spectrometry using API QSTAR™ Pulsar Hybrid LC/MS/MS System.
The protein bands specifically bound to the primary antibodies were detected using an anti-rabbit IgG alkaline phosphatase-linked secondary antibody and an ECF (enhanced chemifluorescence) Western blot analysis system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) (Ouyang et al. 2006).
Some much less serious band was bound to come up with that title, so it was good that Boris claimed it first, as a perfect two-word poem of intent.
WB assays showed that the anti-TvLEGU-1r antibody reacted with the trichomonad 30-kDa band that bound to fixed HeLa cells), suggesting the presence of TvLEGU-1.
The protein band, specifically bound to the primary antibody, was detected using an anti-rabbit IgG-alkaline phosphatase-linked antibody and an enhanced chemifluorescence Western blotting system (Amersham Biosciences, Piscataway, NJ).
The protein band, specifically bound to the primary antibody, was detected using an anti-rabbit IgG-alkaline phosphatase (AP)–linked antibody and an electrochemifluorescence (ECF) Western blotting system (Amersham Biosciences, Piscataway, NJ, USA).
These bands represent ribosomes bound to the mRNA because they were not detected when EDTA was added to disrupt ribosomes by chelating Mg2+ ions (lane 4).
Preincubation of cells with YK5 prior to addition of the YK5-biotin1 and to the affinity purification step, and also affinity purifications with YK5-biotin1 in physiological buffer conditions (i.e., with 150 mM NaCl or KCl) further substantiate that the majority of the bands are indeed bound to YK5 via Hsp70 and thus are potential Hsp70 interactors.
