Saponin vs Triton X 100

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MsV
MsV's picture
Saponin vs Triton X 100

Afternoon all,
I am currently performing (many, many *sigh*) gentamicin exclusion assays with P. aeruginosa at the end of which I lyse my epithelial cells with triton X 100. I keep coming across papers that use saponin for lysis of cells in gentamicin excl assays. What is the difference between using these two methods?

R Bishop
R Bishop's picture
instead of solubilizing the

instead of solubilizing the plasma membranes of the epithelial cells,Saponin pokes holes in it leaving the underlying structures and proteins in the cell intact.  I used to use it all the time to detect Toxoplasma (parasite) inside epithelial cells since the holes will allow antibodies through into the interior of the cell and the parasitophorous membrane, but leave the parasites in tact. TritonX-100, though a mild detergent actually solubilizes the membranes resulting everything spilling out.
My guess is people use Saponin to keep the bacteria intact to measure the gentamicin in some way, sory I've never done that assay per se.
here's some info from wikipedia that explains a bit why this occurs
One research use of the saponin class of natural products involves their complexation with cholesterol to form pores in cell membrane bilayers, e.g., in red cell (erythrocyte) membranes, where complexation leads to red cell lysis (hemolysis) on intravenous injection.[8] In addition, the amphipathic nature of the class gives them activity as surfactants that can be used to enhance penetration of macromolecules such as proteins through cell membranes.[7] Saponins have also has been used as adjuvants in vaccines.
(http://en.wikipedia.org/wiki/Saponin)

MsV
MsV's picture
R Bishop wrote:

R Bishop wrote:

instead of solubilizing the plasma membranes of the epithelial cells,Saponin pokes holes in it leaving the underlying structures and proteins in the cell intact.  I

So is it just the outer membrane that is permeabolised? What about phagolysosomes do you know if these would be disrupted then?
The triton X doesn't blast open the bacterial cells so it allows you to count the total number of bugs in the supernatant, attached to the cells and those that have been phagocytosed or have invaded the cells. I am just unclear how the internalised bugs are counted using the saponin method if the cells are still relatively intact.

A Timmer
A Timmer's picture
We use 0.025% triton-X to

We use 0.025% triton-X to solubilize our cells for antibiotic exclusion assays. This seems to completely eliminate the mammalian cells, but doesn't affect our bacteria (Group A Strep, Group B Strep, Staph aureus). I would check that your bugs are not susceptible to either method before choosing one, but triton-X works great for us.

R Bishop
R Bishop's picture
It is my understanding that

It is my understanding that the phagolysosomes will have pores in them but remain intact. The saponin essentially makes pores in all membranes.  I've only done immunofluoresence with it. I was able to detect a surface protein on toxoplasma inside its vacuole, so it must work that way.
Can you post a link to the manuscript that use saponin? Maybe we can dissect the methods together.
 
I assume your assay is something like this PloS Article on group A strepto? Perhaps they use saponin as very mild lysis compared to triton?
Killing of GAS by human PMNs was determined as described previously [42], but with slight modification. In brief, PMNs (106) were combined with 107 unopsonized GAS in 96-well plates on ice. Plates were centrifuged at 380 × g for 5 min and incubated at 37 °C for 60 min. PMNs were lysed with 0.1% saponin (20 min on ice), and GAS were plated on THY or blood agar. Colonies were counted and percentage GAS survival was calculated with the equation


The assay measures total number of viable ingested and uningested bacteria.
(Sumby P, Whitney AR, Graviss EA, DeLeo FR, Musser JM (2006) Genome-Wide Analysis of Group A Streptococci Reveals a Mutation That Modulates Global Phenotype and Disease Specificity. PLoS Pathog 2(1): e5. doi:10.1371/journal.ppat.0020005)

chrbusch
chrbusch's picture
Actually this is not a reply

Actually this is not a reply but a question. Has saponin been tried to retrieve antigen sites in fixed tissues?

What concentration would you use?

Arvind Singh Pundir
Arvind Singh Pundir's picture
yes saponin is tried  before

yes saponin is tried  before for antigen retrieval, 30 minutes incubation  in 0.05% saponin in milli-Q at room temperature then wash three times in PBS. it may work well for retrieval of your antigen

for reference go to www.jhc.org/cgi/reprint/49/10/1205
and find in Immunohistochemical Examinations heading

Greven
Greven's picture
I want to stain for nuclear

I want to stain for nuclear proteins. Is Saponin better than Triton X for this purpose? Im also using epithelial cells.
Thanks!

mariwan
mariwan's picture
hi all, 

hi all, 
I am working on Toxoplasma proteomic, actually I want to lyse Toxoplasma with out using chemical that affect the protein-protein interaction (I need native protein interacton), I tried Dounce homogenizer for 30 minutes on ice and then sonication on ice for 1 hour but I checked under microscope i found that almost all of my parasites were still intact. I do not need soluble proteins I need only interacted protein related to membrane and organells, so please can I use saponin to destroy the cell or is it working if I use saponin and then sonication. Does saponin affect protein-protein interaction. Do the pores make by the saponin large enough to allow the cytoplasmic soluble proteins to escape out of the toxoplasma or destroy the parasites and permeablise the dodecylmaltoside (unionic detergent to enter the toxoplasma through the pores). 
the protocol
1- Destroy cells and centrfuge to get rid of the supernatant and collect the sedement.
2- Use dodescyl maltoside to solubilizesedement including the membrane related proteins and organelles
3- centrfuge and collect the supernatant and discard the precipitant
in advance, thanks for you paticipation

C. Pardo
C. Pardo's picture
How many time with Triton 0

How many time with Triton 0,025% is necesary to lysate DC´s in Gentamicin protection assay with Streptococcus???, I did the assay in several times but don´t recover any CFU...