SDS-PAGE of secreted protein - (Jan/11/2008 )
My teacher ask me to do SDS-PAGE and our samples are cell culture media
I wonder if we want to study the amount of collagen type I secreted from cell line...What can I do?
1. Can I only collect media. then mix with SDS and then run on the gel? Or I have to do anything else.
2. What % gel I have to prepare. Could you please help me to find proper recipe?
3. Can I use TBE buffer or I must prepare Tris-HCl like common protocol
4. Can I run normal (nondenaturing) PAGE?
Thank you so much...I have never do this before so please help me
well the first question depends of the concentration of the collagen in your medium but i guess you'll need to concentrate it a litel and at least filter the medium for cell debris and stuff. gel percentage depends basically of the molecular weight of the protein. I can't remeber it but 10% should be fine.
for running the sds gel normally it's a tris glycine sds gel. see recipe in maniatis.
i think it wouldn't appropriate to run a non denaturing gel at first because you'll propably see multiples bands so it will be hard to conclude
a lot of questions.... so, let's begin:
1. what are you gonna do with your gel ? stain with coomassie or silver ? western blot ? if you don't have enough protein in your medium, you will have to precipitate proteins... dissolve your precipitated protein-pellet in Lämmli buffer (you need a loading buffer with SDS, a tracking dye like brompehnol-blue, glycerol to let your sample sink into the slot and for denaturating gels beta-mercaptoethanol in pH 6,8)
4. use a denaturating gel....
2. depends on the size of your protein.... 12,5 - 15 % are commonly used....
3. TBE for what ? as running buffer ? you should use a buffer system with Tris, glycine and SDS...
YOU SHOULD READ CAREFULLY A LÄMMLI PROTOCOL::::::
Thank you so much for all of your answers...These help me a lot to begin my work
I only want to study about the amount of secreted protein compare between treated or untreated herb
by using SDS-PAGE followed by Coomassie Blue staining (I do not do Western blot analysis)
I wonder if I add only sds, not add 2-mercaptoethanol, Is there anything change if I do this?
Thank you in advance for all of your advice
are you sure that your collagen of interest is one of the major proteins secreted by your cells ? what kind of cell culture medium do you use ? you should use medium depleted og serum, otherwise you will see mainly serum bans in your coomassie stained gel.
Think about using an antibody for quantification....
Beta-mercapto is needed for denaturating conditions. SDS is only needed for givin your cells a negative net charge to separate them by molecular weight...
Thank you so much, Markusda
My samples are collected from media that have no serum
so I think this does not interfere the results
But I want to know if I don't use coomassie blue
Can I use methelene blue instead?
and I have to centrifuge or filtered my media before running?
i don't think that you can show your collagen by coomassie staining. do you have any idea about the percentage of your collagen in the whole culture medium ? you will see a lot of more abundant secreted proteins but not collagen i think. (correct me if i am wrong and collageen is one of the major secreted proteins in your cells!)
For coomassie staining, you need at least 100 µg of protein. If your collagen is one of the major secreted proteins, measure protein amount with bradford assay and see, how much protein you have in one µl. If it is not sufficient, use TCA or aceton to precipitate protein from the medium an centrifuge after precipitation. dissolve the pellet in lämmli buffer.
My personal advice:
- order an antibody
- measure protein content of your cell culture medium
- precipitate proteins with TCA from sufficient medium to get at least 100 µg
- dissolve proteins in buffer (Tris Buffer e.g.) to a concentration of 5 - 10 µg/µl
- measure exact protein content
- add Lämmli
- load 100 µg whole protein (depending on the quality of your antibody)
- SDS gel
- western blot
I think that you should ask your teacher about the purpose of the experiment, first thing. Our advices for you right now is just 'shooting in the dark'. Depend on the purpose of your exp, the material you start with, there will be certain things that you may or may not need to do. What is more, if you are working with a teacher, the best is doing thing his/her way first, so that any problem you may get, they can help you troubleshoot. Only when the troubleshooting does not work, then you try different ways from other people. It helps not to step on someone's toe.
i completely agree
Hi, I know this might be too late for your work, but in case other people face the same problem as you in future, this is what you should do.
To compare the amount of collagen in different samples of culture medium:
1. To a given volume of culture medium, add 1/10th of volume of 1mg/ml of pepsin in 1N HCl
(pepsin will digest all proteins in the culture medium, except collagen, because collagen's triple helical structure is resistant to pepsin)
Treat with pepsin for 2 hours (with shaking to mix well, but this is optional), then neutralize with 1N NaOH (same vol as the HCl). If your culture medium contains phenol, the colour change helps you to judge if enough NaOH is added.
2. Analyze the samples by SDS-PAGE under non-reducing conditions (reducing or not will give the same results). 3% stacking gel and 5% resolving gel is good enough.
3. The gel can then be stained either with Coomassie Blue or Silver Staining kits. If I understand correctly, Coomassie will give good staining if your sample contains a large amount of the protein, Silver Stain has a better limit of detection, so low quantities of collagen in your sample is easily detected.
That's how it's done in our lab, this is quite routine work for us as we specialize in collagen secretion from fibroblasts. We have detected Collagen I, III and V before using this method.
For more details: Lareu FEBS Letters 581:2709-14 (2007); Lareu Tiss Eng 13(2):385-91 (2007) (Sorry, is this not allowed?)