11 replies to this topic

[cm13]

Hi. I'm investigating a protein that ive found present in my cells.
It reduces when i treat them, and i want to see if its released from these treated cells.

Does anyone have a protocol on how i would go about loading a western blot, using the media that the treated cells are in?
(id imagine i couldnt just load it straight in, considering id have about 5 or 6mls. should i need some way of precipitating protein out of it?)

[mdfenko]

does the medium have serum in it?

if not then you can tca precipitate the proteins or filter concentrate (centricon, amicon, etc).

if there is then the serum proteins will also concentrate. you can try immunoprecipitating your protein.

[cm13]

The media supplement has 0.02 ml / ml  Fetal Calf Serum.
How would this make a difference if im just looking to detect the presence of the desired protein using a western?

Also, the antibody i have for detection is incompatible with the immunoprecipitation kit. So that mightnt be a viable way of doing things.

[vegeta]

Just run the media on the gel and do the western like you would do with cell lysates. I just take the media and run a western on them. There isn't a lot of protein in the media but I have never had a western on conditioned media which didn't give me a signal for what I was looking for.

[lab rat]

You could also enrich your sample through centrifugation. We routinely ultra-centrifuged our cell media to try and concentrate a rare protein from our cell cultures.

[cm13]

yeah, i'd have like 6mls of media to feed the cells. But each well is only 30ul deep.
Would i be better off then centrifuging it, and at what speeds?

[Kaioshin]

cm13, on 15 January 2011 - 02:27 AM, said:

yeah, i'd have like 6mls of media to feed the cells. But each well is only 30ul deep.
Would i be better off then centrifuging it, and at what speeds?

As suggested in the first reply, your best bet is probably TCA precipitation, just google it and I'm sure you'll find a protocol, I don't believe it is terribly difficult.

[mdfenko]

cm13, on 13 January 2011 - 12:21 PM, said:

The media supplement has 0.02 ml / ml  Fetal Calf Serum.
How would this make a difference if i'm just looking to detect the presence of the desired protein using a western?

serum contains a lot of protein. if you concentrate then you may overload your gel. this can cause distortion of the lanes and can affect apparent mobility.

also, if your protein is of a weight similar to the major proteins of the serum then your protein may be obscured.

[cm13]

Protein is about 90kda.
Think the main serum proteins are at 70? is that right?

At present im thinking i should cenrifuge the media sample (at what speeds anyone?)
And then use 20-30ul of the pellet in an SDS gel.

If i do a coomassie blue stain, would that show my protein, or would i have to go ahead and run the western (transfer + antibody incubation, etc) ?

[mdfenko]

cm13, on 19 January 2011 - 08:39 AM, said:

Protein is about 90kda.
Think the main serum proteins are at 70? is that right?

which is close enough to distort your protein if overloaded.

Quote

At present im thinking i should centrifuge the media sample (at what speeds anyone?)
And then use 20-30ul of the pellet in an SDS gel.

there is a calculation for "clearing time" of a protein by ultracentrifugation (it may work in a superspeed but may take a long time). it is dependent on the sedimentation coefficient of your protein and the rcf at which you spin (i can give you the calculation, if you really want it, it's in the beckman ultracentrifuge log book). you could try to pellet the protein, blindly, by spinning at >100 000xg for a few hours.

Quote

If i do a coomassie blue stain, would that show my protein, or would i have to go ahead and run the western (transfer + antibody incubation, etc) ?

yes, you can stain the gel (with coomassie or silver, depending on the protein load). you should be able to make out the band of your protein if it is in sufficient quantity and not obscured by the serum proteins.

[cm13]

mdfenko, on 19 January 2011 - 09:31 AM, said:

cm13, on 19 January 2011 - 08:39 AM, said:

Protein is about 90kda.
Think the main serum proteins are at 70? is that right?

which is close enough to distort your protein if overloaded.

Quote

At present im thinking i should centrifuge the media sample (at what speeds anyone?)
And then use 20-30ul of the pellet in an SDS gel.

there is a calculation for "clearing time" of a protein by ultracentrifugation (it may work in a superspeed but may take a long time). it is dependent on the sedimentation coefficient of your protein and the rcf at which you spin (i can give you the calculation, if you really want it, it's in the beckman ultracentrifuge log book). you could try to pellet the protein, blindly, by spinning at >100 000xg for a few hours.

Quote

If i do a coomassie blue stain, would that show my protein, or would i have to go ahead and run the western (transfer + antibody incubation, etc) ?

yes, you can stain the gel (with coomassie or silver, depending on the protein load). you should be able to make out the band of your protein if it is in sufficient quantity and not obscured by the serum proteins.

I talked with my supervisor - He said it's that im trying to get the microparticles from the cell media and investigate if the protein is presnet in them. (Endothelial cell media. cant remember if i mentioned that)
So i've to pre-spin the media, and then spin it at 20000g at 4C for i think 10 minutes [If anyone wants to suggest better ways of extracting these microparticles, please do]
And then i'll run the pellet on a western under normal conditions.

Does this seem right? Whats the best way to extract these microparticles?

[mdfenko]

yes, it should work. suspend in loading buffer and heat.