I have a query on BrdU/ BrUTP incorporation in culture cells.
Why is transfection/microenjection required for BrUTP incorporation. Whereas, We add BrdU directly into the culture medium to pulse the cells for BrdU.
Is it because BrUTP has 3 phosphate groups and making the molecule bulky?
and it would be highly appreciable, if you can share BrUTP incorporation protocol other than transfection and microinjection.
Thank you
A)yes; it is bulky B) there are no transporters for phosporylated nucleotides and nucleosides and C9) it is negtively charged
Hi, Ashish, I guess that can not load BrUTP inside cells because of it chemical properties. This compond can not penetrate living cell mebrane without transfection, liposome or injection directly into cells. Just compare structure and chemical porperties of BrDU and BrUTP. So, you have to use tretament which made membrane permeable to BrUTP by changes charge and lipohylic properties.
Hi, Ashish,
plesae, consider the chemical properties of BrDU and BrUTP. Second compound can not penetarte membrane of the living cells. So, you have to use tranfection, or liposomes or microinjection,
Only these techniques allow you to penetrate BrUTP and to study level of it incorporation.
For the cell cycle study you can alos use EdU, whiich even does not require antibody for detection. So, you can detect it even much easy to compare with BrDU and you do nit need transfection.
Hi, Ashish beside the reality of it is bulky. i want to clarify that,
BrdU is a synthetic nucleoside that is an analog of thymidine and addition of BrdU to culture medium facilitate their incorporation into the the DNA of cells that are in the S-phase of the cell cycle (no need for microinjection), allow very few BrdU-containing cells to progress through M-phase. This will minimize the number of labeled ‘double nuclei’ and will permit a more accurate quantification of cell proliferation.
BrUTP is used to measure transcription via labeling of ribonucleic acids (RNA). RNA or cells labeled via 5-BrUTP incorporation may be detected immunologically with antibodies. This require transfection and microinjection technique for introduction of BrUTP to permit the observation of nascent rRNA transport while cell permeabilization or microinjection techniques that employed to ensure the intracellular penetration of BrdU allowed the visualization of nucleolar labeling in the vicinity of the polymerase and not permit permit the observation of nascent rRNA transport.
for the protocol of BrdU Labeling of Proliferating Cells see link
regards
courses.mbl.edu/.../protocols/brdu/brdu_protocol_warc
Thank you everyone for clearing my doubts on BrdU and BrUTP. I shall again request you to share protocols for BrUTP incorporation, other than microinjection and lipofection.
Regards.
Hi Ashish Kumar, if you still need a protocol, see this paper, where they used hypotonic buffer to allow the entrance of BrUTP into cells and which could be subsequently labeled with BrdU antibody to detect nucleolar RNA - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977535/
Ehab El-Nahas and Ashish,
The link is not working: courses.mbl.edu/.../protocols/brdu/brdu_protocol_warc
Did you try the BrUTP labeling of the RNA? Will you please share your protocol? I am looking for transfection based protocol.
Thank you very much.
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