Collagen related question

[Maria Semiannikova]

Hi guys, 

I think this question is more to the authors of the paper, but feel free to answer if you have some knowledge. 

What is the reason for diluting collagen IV with collagen I (lyophalized/resuspended)? Because the same final collagen concentrations can be achieved by increasing the collagen I (liquid form) amount and resuspending the collagen IV in a smaller volume than 5ml. And this way all the proportions of collagen I and collagen IV and matrigel will be maintained. Or does collagen I somehow help the collagen IV dissolve? Or will it not reconstitute as well in a smaller volume/higher concentration?

Thanks!

Maria 

[Abdullah Khan]

It's a left over from before I had optimised the diluent. It was very hard to dilute Col IV so you wound up with a low concentration. Since we started using the sodium acetate/acetic acid buffer, you can resuspend Col IV at much higher concentrations so you can do something like what you describe if you want to try it as long as your final amounts remain the same. 

Without the sodium acetate/acetic acid buffer you get basically are limited to a low concentration + high volume, but you can dilute pretty well down to something like 5mg/mL in the optimised buffer. Easy enough to test! Just add a mL of buffer to the Col IV overnight and see if it is enough to give you a decent suspension. 

Abs

[Maria Semiannikova]

Hi Abs, 

Thanks for getting back to me so quickly. Ok, that's great I might try that out. How would I know if it has been resuspended well enough? This is the first time I am working with lyophalized collagen because I have only ever worked with already bought solutions so what are some signs I should look out for? 

Maria