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Those lacking the complete N-terminus of the FH1 domain showed comparatively weak binding to both baits (S440, S433).
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We confirmed binding to both Dynamin1 and Dynamin2 by using GST-tagged GRAF1 SH3 domain as bait in pull-down experiments against purified Dynamin1, brain cytosol, or HeLa cell lysate.
Since all of these contained parts of or the entire DIM region, it seems feasible that SmDia dimerization competed with the binding to the SmTK3 baits.
Competition for binding will also be expected in cases where the prey is endogenously expressed within the Golgi and capable of competing with the LOC fusion protein for binding to the bait-CAT fusion protein.
In principle, since this experiment is based on affinity selection, stronger binding to bait proteins should correspond to a higher probability of detection.
The sieving capability of the NIPAm shell shields the core and its affinity bait groups from larger molecules that may be present and could compete with the intended low-abundance, low molecular weight molecular targets for binding to the affinity bait in the core.
Using pull-down assays with GST-plakoglobin as bait, pPS1 showed lower binding to this protein than npPS1 (Figure 2A).
They are not binding to either party and are open to input from both sides.
Initially GST-DH, GST-PH, and GST-DHPH proteins were used as bait to screen binding, in trans, to both each SH3 domain individually and all five SH3 domains together.
In a quantitative and high-resolution format it enables proteins binding specifically to various types of bait molecules to be distinguished from background binders (Vermeulen et al, 2008).
The yeast two-hybrid system was used with LET-756 fused to LexA DNA binding domain as bait (pDB) for the screening of a human placenta cDNA library containing the Gal-4 activating domain (pAD).
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