What is the mode of action on the following antibiotics: Blasticidin, Geneticin® (G418), Hygromycin, and Zeocin™?

Product FAQ

Answer

Blasticidin: Nucleoside Inhibits protein synthesis in prokaryotic and eukaryotic cells by interfering with peptidyl transfer reaction of protein synthesis, causing early termination of translation.

Geneticin® (G418): Aminoglycoside Blocks protein synthesis in mammalian cells by interfering with ribosomal function.

Hygromycin: Aminocyclitol Inhibits protein synthesis by disrupting translocation and promoting mistranslation.

Zeocin™: Intercalates with DNA and cleaves it.

Answer Id: E3693

Was this answer helpful?

Yes
No
930bb9e689203845528729313d3a2286_FAQ

What is your recommended concentration and solvent for dissolving Hygromycin B? How should it be stored?

Product FAQ

Answer

Hygromycin B is prepared in water and filter sterilized. We used to provide pre-diluted solutions at a concentration of 100 mg/mL, but our current product is provided at 50 mg/mL in a 20mL aliquot.  It should be stored at 2-8 degrees C and should be stable for 6 months to one year at -20C.

Answer Id: E3695

Was this answer helpful?

Yes
No
5c6451fc780eb59e270551d96ebadd64_FAQ

What are the recommended concentrations of antibiotics to use for selection in prokaryotes and eukaryotes?

Product FAQ

Answer

For best results, optimal concentrations for selection should be determined empirically in each unique experiment through dose response curves. However, to get a general idea of concentrations that have worked for individual cell types, please click on the following url: http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/transfection/selection.html or type in “Selection Antibiotics” into our main search on www.lifetechnologies.com.

Answer Id: E3701

Was this answer helpful?

Yes
No
b5ff8626d6afae9110e0d47e8142fc4d_FAQ

Which of Invitrogen™'s antibiotics (Geneticin®, Zeocin™, Hygromycin, and Blasticidin) can be used together for selection in mammalian cells?

Product FAQ

Answer

All of Invitrogen™'s antibiotics (Geneticin, Zeocin™, Hygromycin, and Blasticidin) can be used together. However, kill curves will need to be determined for each combination of drugs since sensitivity to a given drug tends to increase when combined with other drugs.
Note: Puromycin is of the same family as Blasticidin so Puromycin and Blasticidin may not be compatible.

Answer Id: E3948

Was this answer helpful?

Yes
No
8e441594d47e26b35930de3e97f92c07_FAQ

What is the range of specific activity of Hygromycin B?

Product FAQ

Answer

The specific activity range of Hygromycin B is from 386-482 micrograms/mg.

Answer Id: E3696

Was this answer helpful?

Yes
No
b93d4b6ef858bb0781ee51cead07c5cb_FAQ

In contrast to Geneticin® (G418)-induced cell death, cells treated with Zeocin™ do not always detach and float when they die. Is this typical?

Product FAQ

Answer

It is true that a percentage of non-resistant mammalian cells do not round-up from the plate upon Zeocin™ selection as would be seen with G418 or Hygromycin selection. However, one should see some very characteristic morphological changes occurring in those cells that are not resistant. These cells that stick to the culture dish typically display a vast increase in size. This could be best described as being similar to the effects of cytomegalovirus infecting permissive cells. The shape of these cells may also change; taking on an "alien" shape. On close examination of the non-resistant cells, the researcher should observe a distinct breakdown of both the nuclear and plasma membranes. Even though the "cells" are still attached to the plate, they should have the appearance of many holes in these membranes. Also, before the breakdown of the membranes, one can observe open areas in the cytoplasm of the cells that appear to be large, empty vesicles. Although not confirmed, this may be explained by a breakdown of the endoplasmic reticulum and Golgi apparatus, or other scaffolding proteins. Eventually, these "cells" will completely breakdown so that only "strings" of protein are left.

In contrast, Zeocin™ resistant cells should continue to divide at a regular interval to form distinct clumps of cells, or colonies. There should not be a distinct change in morphology, which can be compared to cells not under selection with Zeocin™. It is these colonies of actively dividing cells that contain the resistance gene and are expressing it actively.

If there is concern about the dead cells sticking to the plate, one may do the following to eliminate them: Treat the plate for a couple of minutes with trypsin/versene. Both the healthy resistant cells and the dead cells will dislodge from the plate. The cells can then be replated (without Zeocin™ selection) and the healthy cells will attach again while the dead ones will not. After a couple of hours when the healthy cells have attached to the substrate again, Zeocin™ can be added back to the medium.

Answer Id: E3699

Was this answer helpful?

Yes
No
9d7a8e2d06bcf2e3a0268fad918c7cc3_FAQ