Hey Hector, for cells are you trying to get the collagen and elastin that is in the cell or incorporated into the ECM?  Also, how are you denaturing your samples?

Im looking for ECM proteins Mariya. Im denaturing them with DMSO in the sample buffer and boiling the samples at 100ºC for 5 min.

Well you're right to think that they might need another step.  So the three things I can think of (other than the antibodies but I'm assuming they are previously tested for WB) are:  1) Your denaturation step , 2) Your proteins are stuck at the top of the well, I've seen this with WB for insoluble ECM, 3) Your ECM is still largely stuck on the plate.   Elastin is fairly small, but Collagen I and III are large and once everything is cross linked, it's like trying to get a gum ball to run into your gel.  You might check if you can see a dark line at the top of the wells on your films, that could tell you that protein is getting stuck.  RIPA buffer usually has 1% SDS and that should be doing the trick so maybe it's your denaturing step. They really, really, want to stay crosslinked and depending on how long you are culturing your cells, the collagen might be in big fibrils.  Change your denaturing step to 10% beta-Merceptoethanol for 10 minutes at 90-100.  Same thing for the tissue, although I'm not sure what "incubated the samples for 2 hours at 4 degrees" means?  To your sample buffer add some DNAse.  This will really help loosen up your samples.  Switch to 4-25% gradient gels to help get your collagen in but keep your elastin from running off.  Finally, you might be trying to run too much protein, so you're not getting good solubility.  If you really only need the ECM, try hypotonically lysing your cells from the plate first, then two washes of PBS and then the RIPA.  It will cut down on the protein you have, but should leave the insoluble matrix for your collection.  ---Hope that helps.  I have done a lot of collagen WB and they can be tricky but the nice thing is that the protein can take a pretty good beating and still come up.

Thank you very much! I will try some of those tricks next time. When I said "Incubated for 2 hours at 4ºC" I mean that I left them with the extraction buffer at 4 degrees in  a shaker for that time.

I will update my results.

Thanks! 

Hello Liske,

Yes, finally I made it working. For the extraction I just scrap the cells (grown on a t75 flask) after 2 washes with cold PBS. I scrap them after adding 1 ml of RIPPA buffer (with PMSF, NaF, Protease inhibitor and phosphatase inhibitor). Later I transfer the scrapped solution to an eppendorf (with low protein binding) and I leave it shaking at 4ºC for 2 h.  To finish, centrifuge the cells solution at 14.000 rpm, 4ºC for 15 min. So the supernatant contains the concentrated extracted protein.

I boil the cells at 95ºC  with b-mercaptoethanol for 5 min just before charging the samples in the gel.  For the western at the beginning I obtained too many binds and unespecific antibody binding, so play and try with the primary antibody concentration and the percentage of TBST. 

Hope it's useful for you! 

Hi Hector,

May I ask which antibody you use for the Collagen-I western. We have tried several and all give quite some cross-reactivity with other collagens.

Hello Bram,

We use COL1A2 (G4) from SantaCruz. We had some cross-reactivity, but we  solved that by increasing the amount of Tween in the TBST during the whole process.

Hope it helps you.

Hi Hector,

Thanks for your information. I will give it a try.

Hi Bram

I've been using Abcam's monoclonal anti Col1A1 antibody (cat# ab138492) to detect Col1A1 in cell lysates and spent media of fibroblasts that have been stimulated with TGFbeta. I use a 1:5,000 dilution of this Ab, and a 1:10,000 dilution of the secondary HRP-labeled anti-rabbit Ab from Santa Cruz.

Hi Hector,

I am trying to detect Collagen I from my treated cells. Tried four different antibodies and all the ways people mentioned above, no Collagen detected. I am seriously thinking could be the collagen stuck on the culture plate and was not into the lysis buffer. Do you have any experience on this? 

Thanks a lot!

Kathy

Dear Kathy,

Recently I used some of Santa Cruz's antibodies to detect collagen type I from fibroblast cultures which worked very well. Below you may find the protocol and a representative blot.

-Human dermal fibroblasts cultured in MEM for 72 hours (0.5% FBS, 0.17 mM ascorbic acid, 5 ng/mL TGFb1). Please note that in order to ensure proper production of collagens, you need to add vitamin C to your culture medium, as it is a vital substrate for both prolyl-4-hydroxylase and lysyl-hydroxylase enzymes.

-Cells were lysed in RIPA buffer with added protease inhibitors and the deposited ECM was scraped off the dish with a policeman.

-30µg lysate was denatured for 5 min at 75C with added DTT (10mM) as reducing agent.

-proteins separated on a 6% SDS gel at 100V for 120 minutes using standard Tris/glycine/SDS transfer buffer. 

-proteins were transfered to a nitrocellulose membrane using the semi-dry method for 30 minutes.

-antibodies used: SC-8783 (col-I 1:4000) and SC-8782 (pro-col-I 1:1000).

-The blot shows several lanes of both antibodies used in two different concentrations. The SC-8783 antibody shows three major bands for collagen I (130, 170 and 270 kD). These are the monomer (130 kd), the pro-collagen (170 kD) and the dimer (270 kD). It is even possible to detect a trimeric form of about 400 kD.

-The SC-8782 antibody shows a dominant band for pro-collagen (170 kD). 

-The other fainter bands may be degradation products or unspecific binding.

I hope this helps a bit :)

Good luck!

Hello Kathy,

I had finally results with this conditions, so I summerize them as optional choice to Bram Conditions:

- HDF cultured for 4-2 weeks  in DMEM + FBS 10%.

- Cold extractiton with RIPPA buffer (cell scrapping). 

- SDS Page 10%  with b-mercaptoethanol, after boiling for 5 min at 95º. 5-10 um per well. 120V.

- wet transfer OVERNIGHT at 4ºC (PVDF membrane), 15V.

- Col1A2 (santacruz). 1:600 for blotting.

Good luck!! 

Hello Hector !

I'm actually doing my internship of 2nd year of Master's Degree, and i'm working on the ECM of HDF.

We're having great results with a protocole nearly identical to yours (i read all the stuff you went through, it was helpful). But i'm having quite a bit of a problem, there's a band appearing in almost every WB or Gel, around 55-60 kDa, no matter the Antibody, and i have absolutely no idea what i might be. The results from the LC-MS aren't back yet so i still can't try to find what it is.

By any chance, might you have any idea of what it might be ?

Here's some pics of my gels or WB (Coll 3 and Histone).

Thanks a lot,

Kévin.

MW of Collagen Type I precursor: 140-210 kDa.

MW of mature Collagen Type I: 70-90 kDa.

Collagen I is large and once cross linked, it's like trying to get a gum ball to run into your gel.

Hector Capella how much did you increase your Tween to?

my collagen blots are coming out streaky with Santacruz Col1A, any ideas why?

used RIPA buffer to isolate, 10% gel. Thanks

Hello

I am trying to detect COL2A in vitreous humour of eye. I am using santa cruz antibody SC52658.

WIth non-reducing conditions sometimes i am getting signal but too faint to cal them as bands.

Can anyone suggest me where i could be wrong or what step i need to optimise for the same.