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Blood, Vol. 112, Issue 7, 2738-2749, October 1, 2008
Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation
Blood Hamlett et al.
112: 2738
Supplemental materials for: Hamlett et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 60.9 KB)
- Figure S1. Conceptual basis of antibody blocking used in immunofluorescence experiments to determine co-localisation of proteins (JPG, 59.7 KB)
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(A) Proteins of molecular weight 40KD (e.g. approximate molecular weight of GATA1) and 120KD (approximate weight of FOG1) when represented as idealized red and blue spheres (i.e. in the most compact form) have approximate diameters of 3nm and 5 nm respectively. In reality proteins are not spheres and will occupy a grater volume in space and have longer dimensions. The two-dimensional size of an antibody molecule is 12nm by 6 nm. (B) Schematic representation of two proteins (40KD and 120KD) interacting. An antibody is shown binding one of the proteins (120KD). If a second antibody is added to the experiment, it may be able to bind (top) or may be prevented from binding (bottom panel). In this situation, there is blocking of binding of the second antibody by the first. In general, the degree of blocking is greater the greater the proximity between the proteins. In reality, as proteins are often in multi-protein complexes, rather than in binary complexes, interacting proteins can be more widely separated than shown (making blocking less evident). On the other hand many transcriptional regulators are less than 120KD, thus allowing interacting proteins to be more closely apposed. (C) The extent of blocking of one antibody by another can be quantitated by the reduction in immunofluorescence.
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