MDCK Cells - Science topic

Sure,

Usually the "compactness" is a measure utilized to test how reproducible a method for generating spheroids is. Having a regular shaped spheroid would ensure that test ran on those cells are standarized. Cell density, spheroid size, compacteness and cell viability are all important measures when evaluating the "quality" of a spheroid. Depending on what you are measuring this may be even more important, for example in high throuhput assays for drug testing.

Sadly, I don't have a photo of the spheroids, but in the link I provided you can request access to the paper, which has very clear photos.

I hope this helps

Hi!

It´s a two-step RT-PCR, we use 100ng of cDNA.

The problem may be related to the amount of TPCK-trypsin used in step3 for MDCK cells. The concentration could be too high for the virus inoculation to work properly. A possible solution is to perform a titration experiment to find the optimal concentration of TPCK-trypsin for this procedure.

Dear Michael Essien Sakyi HEK293 cells typically have a higher transfection rate. They are a popular choice for transfection investigations because they have undergone substantial engineering to express high amounts of viral genes. HEK293 cells are a popular choice for transient protein expression and recombinant protein production due to their excellent transfection efficiency with a variety of viral and non-viral vectors.

Thank you for your reply. I will do as you have suggested and hope to get a good result this time. Regards

A few things from what you've written -

You need to consider the exact strain, or at least lineage of "H1N1". Many of the older human isolates of the 1918 lineage H1N1 plaque fine on MDCKs at 37°C. The lab workhorse PR8 for instance. Ditto for the classical swine lineage H1N1s. The same cannot be said for many of the pdm09 lineage H1N1s though, where MDCK-SIATs are needed to match the receptor preference of these viruses. Doing the assays at 35°C for longer also helps (though for us, longer = the 72h you're using).

You also don't say what overlay you're using. We mostly prefer Avicel, though agarose can work well. Carboxy-methyl cellulose will also work, but you can get streaky "comet" shaped plaques which are harder to count.

Final thing that occurs - if the assay is trying to work, titrate the trypsin and/or try an alternative source.

Good luck!

Paul

Two ways to improve titer either by blind passoge or serial passages on SPF eggs

following

Yes we can use it to infect MDCK monolayer with correct moi

Thank you !!

you can use PERMM, my challenge is interpreting the log of coefficient

Thank you..

PCR result may be due to plasmid, seems there is no virus in supernatant. Thanks

Hi, we use MDCK II cells and successfully detect claudins in these cells.

If you use transfer buffer containing SDS, claudins may go through the membrane. I would recommend to omit SDS.

MDCK II cells express claudin-1, claudin-2, claudin-3, claudin-4 and claudin-7. (MDCK I cells do not express claudin-2.) They are all ~22kD and the bands are detected ~18kD in our experimental setup.

For antibody information, see Otani T et al., JCB, 2019 and Saito AC et al., MBoC.

Good luck!

Tomo

Hi Yulin, I'm not sure whether this is helpful or not, but I believe I was having the same problem before during the learning phase of doing the H5N1 plaque assay. Mine was because during performing the assay;

1. I let the cell in the well exposed to the circulating air (without covering with plate cover) for too long. For example, after I took out the PBS from the well (leaving the cell only, without any liquid covering it), I didn't cover the well with its plate cover - letting it exposed to the air while I was taking some media to be put in the same well. Because I was a bit slow before, the cells became dry and died even before I manage to fill in the well with media. So now every time I take out any liquid from the well, I will make sure I only open half of the well surface, and quickly cover the well right after I'm done. I also quickly put the media into the well to make sure the cell isn't being dry for too long.

2. Previously, I also used agarose. Since agarose easily becomes solid, I put it in the water bath while waiting for it to be poured into the well as the last step. The thing was, I didn't realize that the agarose temperature was too high to be poured directly onto the cell. Since then, I changed and started to use the CMC until now (H5N1, H1N1, DENV and CHIKV).

Since I learnt those things in hard ways (I was stuck with the plaque assay for a few months) not realizing my silly mistakes, thus I hope you would be able to overcome your difficulties as soon as possible! Hopefully, my suggestion could help you (if it is the case) :)

All the best, and stay safe! :)

Sha

MDCK cells and did not interfere with IAV adsorptive co_1, also inhibited geenan does not inhibit virus adsorption but inhibits some steps of life cycle inMDCK or A549 cell.

Luciana Tavares Are you using the same culture of MDCK or have you tried starting a fresh culture? I ran into a hiccup with mine because my cells weren't taking to the infection because they were old. (They looked fine, grew fine, but wouldn't infect!)

Thanks Phiyani! I totally agree with that we cannot avoid off-target effect with certain shRNA and it should be compensated by different shRNA targeting same gene. by the way, I'd like to get shRNA sequence targeting my gene of interest, which is predicted to have minimal off-target in dog genome and am wondering which algorithm or website is able to help me with this analysis. Do you have any idea by chance?

First, review the virus. https://viralzone.expasy.org/223

Influenza viruses are enveloped viruses. I would be surprised if you get anything that way. But, what I would do is be very careful with the sediment so you don't mechanically destroy any viruses that are there. Any compression or shearing forces on the sample will almost certainly destroy viruses there.

- So a basic question is, are these existing samples? If so, how were they harvested, how long were the stored and how? Etc..

In the lab, I would dilute the samples in normal saline to try to normalize the forces on the virus envelope. Natively, enveloped viruses are as saline as the cells that bud them off. This makes them absorb water, and causes osmotic bursting.

For sediment samples, i would take small aliquots and very gently agitate them to try to float the virus off. Then I would centrifuge at low speed and remove the supernatant. From there, you could use techniques for harvesting of enveloped particles. Here's some options.

https://www.biaseparations.com/en/featured/viruses

Anything you do is going to tend to wreck influenza viruses. I would suggest that you first culture an enveloped virus in eggs, and learn how to harvest that. You will need to have positive controls anyway. For those, you will need to inject your cultured virus into a sediment or water sample after first slowly adjusting your cultured virus from normal saline aliquot to the level of salinity of your sample.

Before I did all that, what I would do first is send samples out for sequencing. Perhaps you have a sequencing lab already in your university? If so, then do that. That will tell you if there is even any significant RNA influenza RNA in there or not. For that, you will want to use this buffer. https://www.thermofisher.com/us/en/home/brands/product-brand/rnalater.html You can make it yourself too. It will just mean digging around and some more time.

At the worst, you will spend 1 or 2 years learning how to do virus culture, purifications, DNA/RNA sample preparations, assays, and how to use sequencing assembly software.

Since you have ruled out the contamination or mycoplasma infection, you can try to replace the high-glucose/DMEM by MEM/NCS 5%.

hello Alexandra

I used to grow MDCK cells in Minimum Eagle Medium suplemented with 5% GIBCO FBS. Wich kind of plates are you using? It could be another thing to consider if you have no adherence.

Cheers!

I agree with, Michael Theobald

This paper may answer your question.

I did my PhD in a flu lab and remember we often had issues with plaque assays not working! We never fully worked it out but here is some anecdotal advice.

As a gral suggestion we felt like using a relatively 'fresh' batch of MDCKs was important (not too many passages before the assay, ideally only 3-6 splits and no more than 10).

Specifically in your case, maybe the way you are adding the trypsin, directly to the cells, is killing your cells?

One difference to your protocol is that we added the trypsin to the overlay medium. If I remember correctly, the incubation with virus is for these to stick to the cells via HA-sialic acid. Then the overlay medium provides the trypsin needed for HA cleavage and infectivity - but don't quote me on that... it's been a while!

We never suspected about the trypsin and we got ours from Worthington (ScimaR), 10mg dissolved in 100mL PBS and then 0.45um filtered in a sterile hood (store at -80). We added 200uL of Trypsin to a 100mL of overlay medium.

I hope this helps. Good luck!!!!

What strain of H1N1 and what species of RBCs? Not all flu strains have the right sialic acid preference for a given type of RBC. Also, the HA assay is probably the least sensitive routine titration method for flu. If you used an infectivity-based assay you might find you have some replication.

paul

Try using an MDCK-specific transfection reagent (https://altogen.com/product/mdck-transfection-reagent-kidney-cells-ccl-34/) that should help you overcome barriers in efficiency. Because MDCK is not a human cell line (it's canine I believe) generic reagents will not be optimized for the particular cellular markers present on the cells.

I would suggest to adapt the cells fist and select the highest recombinant protein expression second. The cells may lose or change their ability to produce the recombinant protein after the adaptation. Good luck

find the below attachment

Procedure 1. One day previous to infection, prepare 48-well dishes by seeding each well with 7 x 104 cells in 0.5 ml DMEM plus 7.5% fetal bovine serum, 4 mM glutamine, antibiotics. Alternatively another cell density may be required, based on the cell line required for viral growth. 2. On the day of infection, make dilutions of virus sample in PBS. For influenza virus, use cell media plus TPCK trypsin for dilutions. - Tips used for virus transfers: Aerosol resistant 20 μl, 160ul and 1000 μl sterile tips. 3. Make a series of dilutions at 1:10 of the original virus sample. Fill first tube in series with 2.0 ml of PBS, fill the remaining 6 tubes in series with 1.8 ml of PBS. 4. Vortex virus sample, transfer 20 μl of virus to first tube, vortex, discard tip. 5. With a new tip, transfer 200 μl of first dilution to next tube. Vortex, discard tip. 6. Repeat series of dilutions through the last tube. Additions of virus dilutions to cells: 1. Label lid of 48-well dish by drawing grid lines to delineate quadruplicates and number each grid to correspond to the virus sample and label the rows of the plate for the dilution which will be plated. 2. Include four negative wells on each plate which will not be infected. 3. Remove all but 0.1 ml of media from each well by vacuum aspiration. For influenza virus, monolayers should be rinsed once with serum free media before 0.1 ml infection media is added per well. 4. Starting from the most dilute sample, add 0.1 ml of virus dilution to each of the quadruplicate wells for that dilution. 5. Be careful not to track the pipette tip over the wells, and continue infecting the plate with 0.1 ml of virus dilution per well, infecting four wells per dilution, proceeding backwards through the dilutions. 6. Change the pipette tip when necessary. 7. Allow virus to adsorb to cells at 37o C for 2 hours or at a temperature indicated for the specific virus being titered (some viruses grow better at 34o C, including influenza). 8. After adsorption, remove virus inoculum with vacuum, beginning by sucking the most dilute wells and proceeding backwards to less dilute. Alternatively, leave the virus on the cells. 9. Add 0.5 ml Infection Medium (DMEM, 2% FBS, 4 mM Glutamine, 1X PSF) to each well. Do not touch the wells with the pipette. For influenza virus, add Infection Media without serum, with TPCK trypsin. 10. Place plates at 37o C or 34o C, depending upon instructions and monitor CPE using the inverted microscope over a period of one to four weeks, depending on the cultural characteristics of the virus in question. 11. Record the number of positive and negative wells. 12. Calculate the TCID50 titer using the Excel spreadsheet using below attachment

Marcus,

For my cell culture work, I've cut out the broad-spectrum antibiotics. As long as you're working under sterile conditions, then there's really no need to use antibiotics. Depending on how you plan to do your assay, you could use the transwell format for A-B and B-A flux determinations or take a simple plated assay format and look for intracellular accumulation differences between your MDR1 transfected cells vs. wild-type. The advantage of the smaller plate format are reduced cell burden allowing for more conditions to be tested.

Ron

Check media components and lot numbers of FBS.  We heat inactivate our FBS; which is standard in our lab.  Also, we use a different source of MDCKs than from ATCC since we found that their cells were suboptimal and we believe a mixture.

Thanks Erik for your advice!!

Tran, I believe influenza B is also best grown at 33 deg C instead of the typical 37 deg C for influenza A. Thomas's recommendations are also spot on. I have messaged you a protocol that I have used for propagation of influenza B. Joyce 

Hello,

Although I'm not 100% sure, I can suggest one thing:

Do you know why dead cells are positively stained by PI?

PI itself does not distinguish live and dead cells.  It enters the both live and dead cells by diffusion.

Metabolically active cells can pump out PI cells and can keep the concentration of PI inside the cells very low, whereas dead cells cannot.

If your toxin is "pore-forming", it may lead to larger influx of PI into the cells than cells can pump out, even the cells are alive.

Therefore, "PI positive" will not necessarily means "dead" cells.  In other words, if you are dealing with pore-forming toxins, you cannot use PI for live/dead discrimination.

I hope this helps.

Hi Colin,

Contamination is the most challenging and commonly faced problem for all cell culturists. For identification of contamination, i.e. to be sure if at all there is something contaminating your cell culture you may try these:

1. Try not to discard the spent media but keep it in a plate (may be in a 60mm one), if the media turns yellow or cloudy even then, you know it is contamination! To determine what the contaminating organism is, microscopy is the best way.

2. Fix the cells and stain with DAPI. If you see DAPI staining in small dots outside nucleus, you know that it is staining the DNA of your contaminant.

3. Trypan Blue staining showing small dots with dark outline and lighter inner space indicates presence of contaminants.

4. For simple light microscopy, under 40X magnification, if you see black dots stuck on the plate which doesn't seem to go even if you change plate daily, and also has some kind of brownian motion, its bacterial cells that you are looking at.

To prevent contamination-

1. If you have already identified it to be mycoplasma, then there are many preventive measures which might help you. Like you can use 0.1 micron filter to filter sterilize your media and other stuffs of cell culture.

2. I heard Plasmocin from Invivogen is very effective to combat mycoplasma infection. You can follow the link for further information.

3. Also, even though your cells look healthy, their morphology or growth rate doesn't change, appearance of black dots indicate that you need to check for contamination. 

4. Colour change of phenol red is not sufficient enough to determine the presence of contaminant, as rate of colour change depends on the confluency and number of cells seeded. 

Hope it helps!

Hi Asma

Many strains of flu A should form good plaques in MDCKs and when they do, it's a lot more sensitive than HA assays (orders of magnitude). What strain of virus are you using? pdm2009 or some more recent H3N2 human strains won't plaque in MDCKs.

Just to be clear though, you're not measuring particles - you're measuring infectious units; not one and the same, as flu A will often show a particle to PFU ratio of ~ 50. The HA assay indirectly measures particles.

Avicel overlay is a good suggestion, followed by agarose. CMC overlays work, but will often give streaky "comet" plaques which can be harder to visualise.

Presumably you know to use serum-free medium with some BSA and trypsin? Titrating the trypsin is always worthwhile.  The other issue that's not always obvious is that not all MDCKs are equal. You want cells that form "cobblestone" monolayers and that take a long time to release with trypsin when you pass them.

Paul

 Dear Wen Su 

Pls. find the attached files, may help in your research.

Regards

Prof. Houda Kawas

I use basically the same procedure but have HEPES buffer (25mM) added into the 2xMEM/agarose mix. I notice you have sodium bicarbonate but no HEPES. This may be deleterious after a few days as the cells use nutrients and become more sensitive to changes in pH. The paper I follow is: Balish et al. 2013. Influenza: propagation, quantification, and storage. Current Protocols in Microbiology.

Good luck!

I was checking the expression of neuraminidase. Thanks Andrea, it finally worked.  :) 

Hi Miguel 

Thank you for your answer. 

Am familiar with the neutral red assay, however I didn't use trypan blue, so that can be an option for me. 

It is too High shear stress to culture MDCK cell at 120rpm in shaker flasks, and this lead cells to death.

HI Pablo,

1.     The base medium for this cell line is Eagle's Minimum Essential Medium (EMEM). To make the complete growth medium, add fetal bovine serum to a final concentration of 10% to the base medium.

2.     It is recommended that MDCK cells not be used for more than 20-25 passages. New stock cells should then be thawed.

http://digitus.itk.ppke.hu/~cseri/edu/Books/Cell%20Biology/Volume%201/Part%20A/Chapter15.pdf                         

3&4.     Under regular culture conditions, MDCK cells develop tight junctions and form monolayers of polarised cells. Confluency is reached after 3-5 days.

Hope this helps little

Yes, it sounds like the problem is with the titre method from the mouse lung as the animals are clearly getting sick. If you're avoiding freeze thawing after homogenization then the other factor that we've found affects titre a lot is the method of homogenization - we used to use hand-held "mixer" devices (with or without glass beads) and would get low titres, whereas the current Qiagen Tissue Lyser II machine gives us 100-fold higher titres from the same experimental set up. Whether this is because it's faster or simply macerates the tissue better (or both) I don't know.

Bottom line - if the animals are losing substantial amounts of weight, your system is basically working.

Paul

Do not suggest using CCL-34 from ATCC, I believe they are a mixed culture.  Also, you can't passage your MDCKs too many times or they become less sensitive to Influenza virus.  Also as Jessica explains, do not allow your cells to become too confluent prior to infection.  You may be able to register to the influenza reagent resource (see link below) and get a fresh batch of CA/07 to amplify in your lab.  Also, I suggest either using MDCK-ATL or MDCK-London, which are available on the website.  If you have any problems, just e-mail me at txr2@cdc.gov and I can try and help you.  Good luck.

would recommend only a few days if stored @4C.  Do not recommend storing at all at -20C, better to use <-70C for longer term storage.  It is natural to lose some virus (~1/2log) for each freeze/thaw cycle.  If virus was grown in eggs, expect less loss of titer than cell culture since high amount of albumin in allantoic fluid which will help stabilize virus.  If you stored at -20C for a while, you can always reamplify another round in eggs to increase your titer and then store @ <-70C

The answer is that it depends on the cell type. For MDCK cells ,I usually plate them at 2.5x10⁵ and then infect the next day. If you figure they divide overnight, then somewhere between 4-5x10⁵ per well for fully confluent MDCK cells.

2 to 2.5% serum is good enough for infection. TPCK provides acidic environment for HA cleavage during infection. It is needed for infection. Also look into how much virus are you using for infection. Dilute the virus 1:1000, 1:500 and then infect. Good luck

Hello,

I hope it will be helpful

Do you have calcium and magnesium in your buffers that you use to dilute the antibodies?  Cell attachment to substrates is highly dependent on these, that's why we use EDTA as well as trypsin for subculture of course.  So if you don't, you could add  CaCl2 or MgCl2 or both.  

By the way, to subculture epithelial cells, they come off more readily and with less damage with 2 rinses in EDTA before adding the EDTA-trypsin mix, as the EDTA also dissociates cadherins and helps the cells to detach from each other.

You can get some more valuable informations by following the links below:-

1. www.oglethorpe.edu/faculty/~k.../Lab1TissueCulture082808.doc

2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728112/

yea he meant subtraction 

This may help

DMEM media formulation ( 3,64 gm NaHCO3/ liter) is for 10 % CO2 incubator.

Buy DMEM powder and add 2,2 g. NaHCO3/liter as for aMEM, MEM, RPMI...

Hello,

You are right about trypsin resistance. You can try wash the monolayer with Hanks solution or PBS before adding trypsin. Moreover, try to incubate at 37 °С and 5 % СО2. After 5-7 min our cells are detached from the flask. 

you can also try RhMKs (Rhesus Monkey Kidney). 

Thomas

Hi,

as Patrick Keeler has written, the best way is to get the culture directly from the ATCC. You may also ask in any National Reference Center on Influenza or in the  WHO Reference Center (in the UK - NIMR - now rebranded as the Crick Worldwide Influenza Centre). How old is your culture (how many passages did you produce)? Sometimes it's sufficient to recover a new portion of cells from liquid nitrogen to improve virus yield.

I'm amazed you got so much viral genome in the supernatant but not surprised at the lack of HA titre. In our hands the Cal7 virus does not grow in normal MDCKs because of their low 2,6 sialic acid levels, but instead needs to be grown in MDCK-SIATs. Even then, it's a poor-grower, best done at 35°C instead of 37°C because it's temperature sensitive. Using this method, we can get measurable HA titres, albeit low compared to better yielding strains. I don't have cell data to hand, but from eggs we'll get 2e3 HAU for Cal7 or Eng195 (a UK prototype strain) whereas PR8 will give 2e10. We use chicken blood, having tested turkey but not found any benefit.  Remember that the qPCR assay is going to be orders of magnitude (10E3 or 10E4) more sensitive that the HA assay too. Growth time depends on the input MOI of course - 2 to 3 days for a multicycle infection is generally about right.

So - get some SIATs, drop the temperature, stick to a consistent low MOI infection protocol and do a timecourse to work out what's best, and (perhaps) change virus. I'm told that more recent pdm2009 isolates grow better in the lab, despite not having an significant antigenic changes.

cheers

paul

As I am a Chinese whose English is not very proficient, I am a little confused about your problem.

You mean that you obtained 09PDM from MDCK and titrated it by rtPCR or MDCK, but it failed to react with the TRBCs, right?

At first, IAV can coagulate the human RBCs effectively, however, the affinities of HRBCs and TRBCs towards IAV are different. So if you just want to use the HRBCs to assess whether or not your supernatant will cause hemagglutination, then I suggest you to try it. In addtion, Chicken's RBCs can also work well.

Nevertheless, if the results of rtPCR and/or IFA indicated that your cell/eggs produced high quantity of IAV but the hemagglutination assay failed, then I think the problem might exist in the process of  the preservation or/and preparation of the 1%  erythrocytes. Did your TRBCs react with other subtypes of IAV？ 

Meanwhile, the RBCs in UK is far more expensive than that in China lol  We just need spend very little money on Chicken RBSs·····

"To all wells, 100 uL of DMEM medium supplemented with 0.6 ug/mL trypsin (TPCK, Sigma: T1426) were added".

Why do you add trypsin in the last step?! Not comprehensible! 

- During your experiment, make sure that cells don't dry up in plate. Drying kills cells

- All reagents, including media, PBS and the drug should warmed to 37 deg C.

- Your plant extract may be toxic. Try lower concentrations, lower incubation time.

- You may remove plant extract after few hours and replace with normal cell culture medium  (MEM+ Bovine fetal serum+L-glutamine).

- Normally,  Trypsin is never added to DMEM during culture! It is only used for detaching cells for splitting or passage.

A549 cell line was established from lung adenocarcinoma tissue and the signal transduction is totally different from normal airway epithelial cells and alveolar epithelial type I and II cells. In particular, A549 is famous for the silencing CD44 expression, which is essential for the binding to ECM components such as hyalronic acids. To better understand the Influenza biology, the glycobiology is essential, so that CD44 expression should be positive in the cell lines in the research of interest.

Jackson ImmunoResearch  has some of the better simple conjugates and has a variety of fluors depending on your needs, I like them because they are a little more cost effective.   I can't give  any advice on a protocol as concentration/timing varies according to your assay.  I have also used the NeutrAvidin binding protein above, it also works. So you are aware that streptavidin is quite large and may have an affect on the cells ability to endocytose your extracellular proteins?

I simply do not know

We studied this adaptation through passaging in our previous paper (Ramadhany et al. 2012 ; Yasugi et al., 2012), when we cultured human influenza viruses in embryonated eggs, some mutations occurred in HA binding site even only after 1 time passaging. The population of viruses carrying mutation will become dominant through passaging in egg. To lessen these mutations, culturing human influenza viruses in MDCK cell might be better option.

Hi Philip, We routinely use fibrillar collagen as a matrix for MDCK II (Heidelberg strain) cells and they adhere just fine mainly via a2b1-integrins. Which MDCK cells are you using? Another issue is the elastic modulus or stiffness of your gel. Have you tried more rigid recipe? From the low magnification images it is difficult to say whether your MDCK cells grow really as monolayer cell islands or maybe they form 3D clusters due to excessively soft PAA-gel? Have you looked at this in more detail?

1 / I get to have the results of whole genome sequencing of different viral strains and I need to know the positions of the mutations and their kind, so is there a bioinformatics software to do this step and how I can the analyzed 

2 / I am trying to construct phylogenetic trees for every gene of influenza virus, so my question is: is that the variability observed on the trees are all real or may be due to the PCR reaction (enzyme)

3 / I want to understand response and antibody epitope knowing that I will soon start a IHA test

MC from Sigma work just fine..

Saturation can happen on several levels. I'm not sure what you are doing, because there are different ways in which co-IPs are done and people have different strategies. If proteins X and Y interact with each other in MDCK cells, you assume that X-GFP still interacts with Y in those cells, then when you make a lysate, you hope that X-GFP continues to interact with Y. So you incubate your centrifuged lysate with anti-GFP antibodies for 1 hour on ice, you spin briefly to remove unspecific protein aggregates, then you mix the supernatant with protein-A sepharose (or magnetic beads) and put it on a slow rotator in the cold room for 2 hours sustained capture by protein A, then you separate the immobilised protein A beads, wash them, separate them again etc.. and then you boil the pellet in sample buffer and run on a gel. How do you detect Y? Have you labelled your cells with 35S methionine and do you expect a radioactive band at a specific molecular weight in addition to the X-GFP band? Or do you have an antibody that specifically detects Y?

If this all makes sense and I'm on the right track, then here is what I think could happen with respect to saturation:

1) There is a limited amount of Y in your cells, so if you overload with X-GFP, and immunoprecipitate only a portion of the overexpressed X-GFP, then you loose a lot of Y interacting with those X-GFPs you failed to precipitate (either due to limiting anti-GFP antibodies or limiting spaces on proteinA beads).

2) In fact you can also overload with primary antibody, imagine you add so much anti-GFP serum that all the X-GFP (and associated Y) is bound to antibodies, but only 10% of those antibodies can be captured by protein A beads, then that will effectively dilute your signal on the proteinA beads by a factor 10. proteinA must be in excess of antibody. Antibody must be in excess of antigen (X-GFP). And X-GFP must be present in a reasonable level relative to protein Y that you expect to interact in vivo.

Ideally, a reviewer will say that you should do reciprocal IP, repeat everything, but switch Y and X around (make Y-GFP and test if it co-precipitates X)

This is my best guess to answer a question on saturation, but I agree with Amanda that your description doesn't specifically state what you do and what your concern is. I still hope the answer helps though.

2 methods:

1) Quick method, following clarification (2200xG, 10min @4C), pellet virus 45,000xG for 3Hr @4C or 103,000xG for 1.5Hr @ 4C.

2) If you want intact virus you can band pelleted virus from method 1 over 30%/60% sucrose step gradient (in Calcium Borate buffer) for ~1.5Hr @50,000xG 4C. Using Calcium borate buffer instead of PBS will help retain virus NA activity.

Pl grow in serum free medium

Where did you get this from ATCC or the from primary cultures from the tissue.

They could be osmophilic, glycogen or zymogen granules....

check these two articles

1.http://www.atsjournals.org/doi/pdf/10.1165/ajrcmb.17.6.2858

2. http://www.nature.com/ki/journal/v78/n1/pdf/ki201068a.pdf

hope this helps.

RKG