How does the treatment cytoplasmic homogenate with N-ethyl maleimide with SDS prior to electrophoresis work?

Question

N-ethyl maleimide (NEM) reacts with free sulfhydryls

If you don't treat with NEM, the gel looks horrible after electrophoresis.

Answer 1

It was postulated that specialized microdomains of the plasma membrane, consistent with caveolae, might play a role in cholesterol trafficking in intestinal cells. The existence, therefore, of caveolin and the role of detergent-resistant microdomains of the plasma membrane in cholesterol trafficking were investigated in human small intestine and CaCo-2 cells. Caveolin mRNA was detected by RT-PCR in small intestinal brushings and biopsies and in CaCo-2 cells. Northern hybridization of caveolin mRNA detected 3 kb and 0.8 kb transcripts in CaCo-2 cells. From brushings of distal duodenum and in CaCo-2 cells, Western analysis for detection of caveolin protein demonstrated a 21 kDa-sized protein and a 600 kDa homooligomer. In CaCo-2 cells, caveolin was demonstrated by immunoflourescence in apical membranes as well as within cells. Using sucrose-density gradients, caveolin was localized to detergent-resistant microdomains of the plasma membrane. As determined by cholesterol oxidase-accessible cholesterol, 3–5% of plasma membrane cholesterol in CaCo-2 cells was estimated to be in these detergent-resistant microdomains. After the absorption of cholesterol from bile-salt micelles, more plasma membrane cholesterol moved to these specialized microdomains within the plasma membrane and was esterified. In CaCo-2 cells, filipin, N-ethyl maleimide, and cholesterol depletion, treatments that disrupt caveolar function, interfered with the transport of plasma membrane cholesterol to the endoplasmic reticulum, whereas okadaic acid, sphingomyelinase, and cholesterol oxidase did not. Changes in cholesterol flux at the apical membrane of the cell did not alter mRNA levels or mass of caveolin.

The results suggest that caveolin is present in intestinal and CaCo-2 cells and is associated with detergent-resistant microdomains of cellular membranes. With the influx of micellar cholesterol from the lumen, plasma membrane cholesterol moves or "clusters" to these microdomains and is transported to the endoplasmic reticulum for esterification and eventual transport. Caveolin/caveolae may play a role in cholesterol trafficking in intestinal cells. It is well recognized that mammalian cells tightly control the amount of unesterified cholesterol they contain. It would seem reasonable to assume, therefore, that after the uptake of additional cholesterol from the gut lumen, the intestinal cell would have an efficient and orderly mechanism for transporting excess cholesterol from the plasma membrane to the endoplasmic reticulum. This would then ready the sterol for secretion (as a part of a lipoprotein particle) or regulate expression of sterol responsive genes, such as HMG-CoA reductase and the low density lipoprotein receptor.

Answer 2

No idea why you'd use NEM, are you doing protein electrophoresis? What is your protocol? Generally for mamallian tissue culture cells, you can remove the media, wash with PBS, and add 1X SDS PAGE sample buffer, which contains 1% SDS and beta mercap-ethanol. This immediately lyses the cells and denatures the proteins. Then, shear the extract with a 23 gauge needle to break up the DNA (otherwise you can't load the sample onto the gel), boil for 5' , and run the gel. You should get good results if you have a confluent 10cm dish, lyse with 100 ul of sample buffer, and load 10 or 20 ul of it.