The nucleoporins are a family of proteins which are the constituent building blo

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The nucleoporins are a family of proteins which are the constituent building blocks of the NPC

2. The nucleoporins are a family of proteins which are the constituent building blocks of the nuclear pore complex (NPC).The nuclear pore complex is a massive structure that extends across the nuclear envelope forming a gateway that regulates the flow of macromolecules between the cell nucleus and cytoplasm. Nuclear pores in turn allow the transport of water-soluble molecules across the nuclear envelope. Nucleoporins (abbreviated Nup) , a family of around 30 proteins, are the main components of the nuclear pore complex in eukaryotic cells In an experiment (see figures A-G), a cell line (HeLa) was treated (0.2 mm and 2 mm) with peroxide (H202) to induce oxidative stress and the nuclei were extracted and proteins separated on a SDS-PAGE gel. The extractions were done using N-ethylmaleimide (NEM) to prevent disulfide formation during preparation and under two conditions one with Dithiotrhreitol (DTT) and one without DTT. Dithiothreitol is a reducing compound. Different antibodies with a fluorescent label was used to detect a specific Nucleporin protein in gels A - F. eg. in A, a- Nup358 will produce a band that is denoted by the asterisk() above 200 kDa. The location of the other Nucleoporins (NuP) are shown (B- F) by varying asterisk in each gel. a. For most of the gels, there is an arrow shown pointing to a band (s) located at higher position. Are these band (s) a greater or lesser size than the nucleoporins designated with the asterisks? b. If the antibody is specific for that particular nucleoporin, then why might it be recognizing a different band? c. Why was dithiothreitol (DTT) used and what do the results with DTT mean? d. Which nucleoporin(s) demonstrate the phenomena represented by questions a-c? For figure “G” a similar experiment was done as before, however the first lane serves as a control and the second lane used a techniques on the cells that prevents the expression of nucleporin 62. Each of the three gels is looking for the presence of three different Nucelporins (Nup358, Nup153 or Nup155). Using what you learned from the answers above, what can be concluded from Figure G? e.

Explanation / Answer

a. The bands pointed by arrows at higher position indicate they are of greater size than the nucleoporins designated with asterisks.

In an electrophoresis, molecules with larger molecular weight migrate slower on gel compared to molecules with smaller molecular weight.

b. an antibody specific for a particular nucleoporin if recognizing a different one, it is due to cross reactivity. When two molecules share a similarity in its antibody binding regions, an antibody particular to one molecule would produce a band for the other molecule too.

c. DTT acts as a reducing agent, i.e, it prevents the formation of disulfide bonds and thus secondary structures in proteins. A secondary structure containing protein, when mixed with DTT, converts into a linear chain of amino acids and hence migrates faster.

d. since the lower bands (marked with an asterisk) are present in all the lanes (-DTT and +DTT), it is difficult to trace the changes. The upper bands are not visible clearly.

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