bochum

For my work i have to do a western blott for 320 KD protein. I tried 7% gel. Bought a gradient gel of 2%-16%. Run the gel overnight (4 degree). Transferred 3hr in cold room. Still not able to detect the protein.

Suggest where am failing.

Thanks

A protein that I immunoprecipitated from cell lysate, runs a little bit higher than the expected size. Has any one observed IP samples running at incorrect molecular weights on a 12% SDS-polyacrylamide gel?

Our genetic material is often compared to a book. However, it is not so much like a novel to be read in one piece, but rather like a cookbook. The cell reads only those recipes which are to be cooked at the moment. The recipes are the genes; 'reading' in the book of the cell means creating RNA copies of individual genes, which will then be translated into proteins.
The cell uses highly complex, sophisticated regulatory mechanisms to make sure that not all genes are read at the same time. Particular gene switches need to be activated and, in addition, there are particular chemical labels in the DNA determining which genes are transcribed into RNA and which others will be inaccessible, i.e. where the book literally remains closed. The biological term for this is epigenetic gene regulation.
Among the epigenetic mechanisms which are well studied is the silencing of genes by methyl groups. This is done by specialized enzymes called methyltransferases which attach methyl labels to particular 'letters' of a gene whereby access to the whole gene is blocked. "One of the great mysteries of modern molecular biology is: How do methyltransferases know where to attach their labels in order to selectively inactivate an individual gene?" says Professor Ingrid Grummt of the German Cancer Research Center (DKFZ).
Grummt has now come much closer towards unraveling this mystery. She has focused on studying those text passages in the genetic material which do not contain any recipes. Nevertheless, these texts are transcribed into RNA molecules in a controlled manner. "These so-called noncoding RNAs do not contain recipes for proteins. They are important regulators in the cell which we are just beginning to understand," says Ingrid Grummt.
In her most recent work, Grummt and her co-workers have shown for the first time that epigenetic regulation and regulation by noncoding RNAs interact. The scientists artificially introduced a noncoding RNA molecule called pRNA into cells. As a result, methyl labels are attached to a particular gene switch so that the genes behind it are not read. The trick is that pRNA exactly matches (is complementary to) the DNA sequence of this gene switch. The investigators found out that pRNA forms a kind of plait, or triple helix, with the two DNA strands in the area of this gene switch. Methyltransferases, in turn, are able to specifically dock to this 'plait' and are thus directed exactly to the place where a gene is to be blocked.
More than half of our genetic material is transcribed into noncoding RNA. This prompts Ingrid Grummt to speculate: "It is very well possible that there are exactly matching noncoding RNA molecules for all genes that are temporarily silenced. This would explain how such a large number of genes can be selectively turned on and off."
Source : Helmholtz Association of German Research Centres

During a pcr when the DNA melts and temperature is brought to 50 and the primer binds the single straned DNA,then why doesnt the original DNA reneal too when the temperate is brought to 50.

The mammalian fucose mutarotase enzyme is known to be involved in incorporating the sugar fucose into protein. Female mice that lack the fucose mutarotase (FucM) gene refuse to let males mount them, and will attempt copulation with other female mice. Researchers writing in BioMed Central's open access journal BMC Genetics created the FucM mouse mutants in order to investigate the role of this enzyme in vivo.
Chankyu Park worked with a team of researchers from the Korea Advanced Institute of Science and Technology and intriguingly gained some insight into the neurological basis of sexual preference. He said, "The FucM knockout mice displayed drastically reduced sexual receptivity, although pregnancy after forced mating attempts by normal sexually experienced males showed that the animals were fertile. The FucM knock-out mice have reduced levels of alpha-fetoprotein, a protein thought to be involved in development of parts of the brain responsible for reproductive behavior".
The mutant female mice were healthy, and behaved normally towards young mice. When approached by male mice, however, they would not adopt the sexually receptive 'lordosis' position. Furthermore, they lost interest in investigating male urine, unlike normal females, and would attempt to mount other females. Speaking about the results, Park said, "We speculate that these behavioural changes are likely to be related to a neurodevelopmental change in pre-optic area of the female mutant brain , becoming similar to that of a normal male".
Source : BioMed Central