6 replies to this topic

[ebnguyen]

Dear all,

I am a total newbie in performing SDS-PAGE and I'll be happy if you could help me on this small problem and yet so troublesome for me! I am trying to see/measure collagen from cell medium and cell monolayers (fibroblasts) by SDS-PAGE.

I get no band with my collagen control with comassie stain. collagen bovine I (tendon achille) from sigma C6897 was dissolved in 0.5M acetic acid with 1mg/ml pepsin for 4 days. I tried to load the collagen solution as it is in the gel, neutralize it or even precipitate it in acetone and redilute in SDS buffer, or PBS. but still, i got no band at all. what could be the issue? collagen no solubilized enough? i am sure I put enough collagen (around 200 ug! instead of 30 ug).

what about collagen extraction from medium and cell layer? do you advise me to use pepsin to digest all proteins except collagen? sonication? triton-X?

i've tried already many different ways and still got new results. I will really appreciate if someone could enlighten me!

thank you for any advise

ebnguyen

----
Protocol I used:
1) Medium samples: precipitate in acetone overnight at 4°C, centrifuge at 4,000 rpm for 10min, resuspend pellet in either SDS buffer or PBS
2) Cell layer samples: incubate cell layer at 4°C for 10 min with 1% Triton-X with protease inhibitor, precipitate in acetone, centrifuge at 4,000 rpm for 10min, resuspend pellet in either SDS buffer or PBS. Or scrap cells in a solution containing protease inhibitor, sonicate and precipitate in acetone, centrifuge at 4,000 rpm for 10min, resuspend pellet in either SDS buffer or PBS

Edited by ebnguyen, 30 May 2012 - 03:42 AM.

[TRY]

same thing i am having problem with since 2 weeks...i am also not getting the bands of collagenase digested collagen i (c8919). ..i did comassie stain..i dont know if acetic acid used during fixation step drains off the collagen in the gel.

[ebnguyen]

I forgot to mention: i am using 12% gel and in reducing condition (mercaptoethanol/boiling). any suggestion? i am out of ideas...

[Arun Kumaran]

Keep everything in cold condition during extraction. Check your protein solubility since solubility differs based on the type of collagen. Acetic acid is a good fixative except for some low molecular weight proteins. For low molecular weight proteins, fixing solution is different. Try to add DTT (Final conc: 50 - 100 mM) before boiling and increasing the percentage of gel.

[ebnguyen]

Thank you Arun Kumaran!

you suggest to increase the % of the gel? I read in some others posts that for high molecular weight protein, I should use a lower % gel (5%?).

[genehunter]

Its been a long time sicne last post here, years, maybe.  Hi, everyone!

ebnguyen:

A number of issues may cause your problem. Collagen in bovine tendon achille is highly cross-linked and very insoluble in water due to post-translation modifications. Your extract condition might not work as efficiently as you think.  Have you measured the protein concentration of the extract? Collagen subunit has high MW, 12% maybe too high. Try 8% or 10% gel instead. 5% only good for HMW assembeled protein complexes.
You asked about the residual organic solvent in samples, did you notice any loss soon after you load your samples into the wells? If so, use dialysis or speedvac to remove it.

[ebnguyen]

Hello Genhunter

thank very much for answering.

for the collagen control, should I use then another collagen (instead of the collage bovine tendon achille which is highly cross-linked). I have not measured the concentration of the extract (which I will do today!).

as for the residual organic solvent in the samples, I do see only a small band of bluish going at the bottom of the well (as for the control marker sample, it fills up the whole well). I don't know if it means my sample is flying away during the loading. but that's what I noticed...

again, thank you for any advice!

Edited by ebnguyen, 02 June 2012 - 08:44 PM.