Since the size of the protein is bigger, you have to run longer time if you are using semi-dry blot(I attained by running 40min at 21Volts; Biorad). Did you check ponseaus stain of your blot? (where you have to see the transfer of total proteins, gives an idea about just transfer). Did you check any Tubulin/Actin staining? (will also be a proof that your technique worked).

I dont really care for blocking (overnight is unnecessary to me) but I do standardize the primary antibody incubation. If I dont see any band after 1 hour incubation, the next step I would do is overnight primary antibody incubation! the next day I wash and probe with secondary Ab for 1h. You may try this.

Start with coomasie gel staining, 0.5ug you will see. If you see it on coomasie, stain the membrane with the Ponceau-S, where you will see it as well. Since you have added 0.5 ug of collagen it is not possible not to detect it with W-B procedure you described. Thus I expect gel/transfer problem, to help you I need the full description of your gel/transfer conditions.

What chemiluminescent reagent are you using? Pico or Femto ECL? Maybe you should try femto and also try to reduce membrane blocking for 1 h and put primary Ab during the night in 4C.

To check your ECL add 0.1ul of the secondary HRP antibody to the 200ul of the solution. In the darkroom you should have with your eppendorf a little lamp lightning...

Hi Dominik,

I am working with lots of different collagens and I really saw that for some reason with this moleculs wet blotting is superior to semi-dry! I normaly blot wet for 3hrs. at 75volts. But Col1A2 is not very large so 1,5hrs at 100V will do. Concerning blocking I would reduce to 1hr. maybe even 2,5% depending on the backgroundsignal you got.

Is the transfer of you ladder complete? This gives a hint that the transfer is working fine.

I did a quick glance at abcam and non of their antibodies required nen-reducing conditions so I would not go there... (and deal with a triplet of 420kd).

What cell line do you use? As Collegens are secreted you might need to block secretion to get a good signal, shifting the cells to 40°C over night works very well!

Best!

Patrik

Hey guys, thanks for the answers, really good input!

- coomassie: col1a2 band could be seen upon coomassie staining

- ponceau S: does not work very well on PVDF; no band could be seen

- ECL: substrate works with the 2nd antibody

- HK: GAPDH detectable

@ patrick

i will definitely try wet blotting this week, thanks for the advice.

transfer of the ladder was complete except for 170 kDa.

i couldn't even detect positive control (0,5µg, purified collagens from millipore/acel)

Remember to have at least 0.1% SDS in your transfer buffer. Without it is very hard to transfer large proteins with semi-dry. Your GAPDH is only 38kDa. What is actually your gel concentration (if over 8% I would be really afraid of the transfer)? Suggest gradient gel 4-15% like TGX of BIORAD I'm using, or equivalent from any other company. Keep fingers cross:)

To me this really looks like a problem related to antibody/blocking rather then transfer. But to be shure I would only run 6% gels (Col 1A1 runs at around 170kd although its real size is only 140kd...). If you want to be sure your antibody and blocking are working and you have the pure protein at hand, you can just spot a little amount on you activated PVDF (Nitrocellulose works better), block it and detect with your antibody.

I have perform analysis of type I, II, IX, and XI without antibodies, using degradation of protein by pepsin and migration of purified collagen. You could see this method in 2 publications.

Article Native and DPPA cross-linked collagen sponges seeded with fe...

"Nitrocellulose works better" as Patrik said, I suggest you a romance with this membrane I’m having already all my adult life. You have only one buffer for transfer, do not need to activate. The capacity of PVDF is not necessary for Western, since when you reach it, it means your gel is overloaded like a hell… This is a good membrane for lazy people I’m:)

I dont think the semy dry transder is the problem. Now im detecting col1a1 (210-140 kDa) with 20 ug of protein in 6% gel using a semy dry transfer.for 2 small gels i set 20 v, 400mA and 1h! Works perfect

Thanks again for all the suggestions that I received - they were of great help!

On the one hand I was able to detect COL1A2 in lysates now, but the positive control (bought human col I) does not work constantly, which is annoying. This is the setup:

- 30 µg of lysates; 0,5 µg of pure Col I

- 6% SDS gels

- elpho: 120V, 90 min

- wet-blotting: 350 mA, 90 min, PVDF (will also try NC)

- blocking: 30 min 5% Milk in PBS-T

- prim AB: 1:500 in 5% BSA/TBST, over night, 4°C

- sek AB: 1:5000 in 5% BSA/TBST, 2 h RT

- detection: ECL

Could i ask you which ab are you using And the size of the specific band you get? Do you get other bands? Thank you in advance.

We're using sc-28655 anti-COL1A2 and sc-8785 anti-pro-COL1A2 antibodies. For anti-COL1A2 we only see one very faint band at ~130 kDa. For the latter we see a very pronounced band for the cleaved propeptide at ~30-40 kDa, a weaker band at around 170 kDa and quite a number in between that we can not assign yet.

Thanks a lot!!

Hello! I know it's been 5 years since this thread received any attention, but Dominik, did you figure it out? If so, which antibody worked best for you?