Buffers - Confused about the compositions (Jun/03/2009 )
little mouse on Jun 4 2009, 06:18 PM said:
jiajia1987 on Jun 4 2009, 11:37 AM said:
Thanks for your reply.
Why would BSA be a better choice than milk?
And where does NaCl come into the washing steps? So far, my washing buffer contains 0.1% (w/v) BSA, 0.1% (v/v) Tween-20 in DPBS. Does NaCl come in the form of a powder? I am sorry if this sounds stupid.
no milk because there is biotin in milk.
DPBS is Dulbecco's PBS. PBS is phosphate buffer saline, with 150 mM NaCl
Buy some NaCl powder from sigma and add it to your solution.
I didnt know that there is biotin in milk, thanks for telling me!
By the way, how is the additional NaCl going to help in the washing step? Thanks once again.
And, I am thinking of making DPBS with BSA for incubation of my beads with the templates. Was wondering if I should add tween into the buffer too? Or just have DPBS with tween for the washing steps.
increasing the concentration of NaCl will disturb the binding of proteins (electrostatical interactions), but you can disturb the binding of biotin too, that's why you should try different concentrations up to 500 mM
You can try tween. actually you have to try several things and always have a control without biotin. if you don't have enough biotin bound, decrease NaCl and/or remove tween, if you have two much background, increase NaCl and/or add tween.
good luck
little mouse on Jun 5 2009, 04:23 PM said:
You can try tween. actually you have to try several things and always have a control without biotin. if you don't have enough biotin bound, decrease NaCl and/or remove tween, if you have two much background, increase NaCl and/or add tween.
good luck
Thanks so much for explaining things out to me. I really appreciate it. Not everyone is helpful here in my lab and I was almost at the verge of crying from having to face attitude problems. Many thanks to you again.
jiajia1987 on Jun 5 2009, 10:25 AM said:
little mouse on Jun 5 2009, 04:23 PM said:
You can try tween. actually you have to try several things and always have a control without biotin. if you don't have enough biotin bound, decrease NaCl and/or remove tween, if you have two much background, increase NaCl and/or add tween.
good luck
Thanks so much for explaining things out to me. I really appreciate it. Not everyone is helpful here in my lab and I was almost at the verge of crying from having to face attitude problems. Many thanks to you again.
You're welcome