Detection of Radiolabelled Proteins

Proteins may be radiolabelled during synthesis in the presence of labelled amino acids. 35S methionine is commonly used in this regard. Figure 4, for instance, shows biosynthetic labelling in human lymphocytes

Figure 4 A 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of 35S methionine-labelled proteins from human lymphocytes after different recovery periods from 1 h heat shock at 42°C. Control cells from the same individual were maintained at 37°C. Lane 1, 2 h control; lane 2, 2 h after heat shock; lane 3, 3-h control; lane 4, 3 h after heat shock; lane 5, 4-h control; lane 6, 4 h after heat shock. Numbers to the right of the gel indicate the position of major heat shock proteins (hsp) induced after heat shock. Hsps are routinely classified according to their apparent molecular weight (105, 90, 70 kDa, etc.) after one-dimensional SDS-PAGE. (Thanks to my PhD student D. Visala Rao for this gel.)

Figure 4 A 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of 35S methionine-labelled proteins from human lymphocytes after different recovery periods from 1 h heat shock at 42°C. Control cells from the same individual were maintained at 37°C. Lane 1, 2 h control; lane 2, 2 h after heat shock; lane 3, 3-h control; lane 4, 3 h after heat shock; lane 5, 4-h control; lane 6, 4 h after heat shock. Numbers to the right of the gel indicate the position of major heat shock proteins (hsp) induced after heat shock. Hsps are routinely classified according to their apparent molecular weight (105, 90, 70 kDa, etc.) after one-dimensional SDS-PAGE. (Thanks to my PhD student D. Visala Rao for this gel.)

amino acid into the particular protein zone but also on the isotope itself. For example after a 24-h exposure about 300-500 d.p.m.cm2 of 32P will give a visible band whereas about tenfold this level of radioactivity is required to produce a visible band using 35S or 14C label. 3H label is not detected because the low ^-emissions do not penetrate the gel matrix. A fiuor may be introduced into the gel matrix prior to gel drying to detect 3H-labelled proteins as well as to improve the sensitivity to 14C and 35S.

Proteins may also be labelled post synthetically using such reagents as 14C iodoacetic acid which preferentially labels available sulfhydryl groups (see Figure 1, insert lane 1) and 125I which preferentially labels available tyrosine groups. Many laboratories use phosphoimaging whereby the radioactive proteins in the gel excite a phosphorescent screen and the number of excitation events is directly digitized. Although this instrumentation is rather expensive it obviates the need for X-ray film and gives results in hours rather than days. Effective concentrations are said to be linear over six orders of magnitude whereas the linear range for X-ray film detection rarely covers one order of magnitude. For further details see the separate article on Detection Techniques: Staining, Autoradiography and Blotting. Radioactivity may also be quantified in gels after slicing and solubiliz-ation. In this case, a generally useful labile crosslinker N,NJ-diallyltartardiamide (DATD) is often used in place of bisacrylamide.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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