Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Buffers


  • Please log in to reply
14 replies to this topic

#1 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 03 June 2009 - 12:45 AM

Hi peeps,

I am going to design an experiment, and I need to come up with blocking buffers. I am new to this so do pardon me if I sound stupid!

I have heard of people using tween in PBST and milk in western blot blocking buffers to prevent non-specific binding. However, how does all these blocking substances (tween or milk for example) work? How should one determine their concentration in the whole buffer? And how do we know what other ingredients can be used to prevent non-specific binding or for more stringent washings?

Thanks in advance!

#2 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 03 June 2009 - 01:23 AM

Hi peeps,

I am going to design an experiment, and I need to come up with blocking buffers. I am new to this so do pardon me if I sound stupid!

I have heard of people using tween in PBST and milk in western blot blocking buffers to prevent non-specific binding. However, how does all these blocking substances (tween or milk for example) work? How should one determine their concentration in the whole buffer? And how do we know what other ingredients can be used to prevent non-specific binding or for more stringent washings?

Thanks in advance!



tween will help to wash out proteins that bound non specifically (weak binding). Milk contains protein (you can also use BSA) that will bind via protein protein interactions, non specifically. Then you will have a competition between milk proteins and your antibody with the non specific binding sites. There is a competition for the specific sites, too, but the antibody will win.
As there is an excess of the blocking proteins, you will mainly have milk proteins bound on non specific sites and antibodies on specific sites.
Usually we use up to 5% non fat dry milk or 1% BSA.
You have to try which one is the best, and try different concentrations, increasing if you have background, decreasing if you don't have enough signal.

#3 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 03 June 2009 - 05:24 PM

Hi peeps,

I am going to design an experiment, and I need to come up with blocking buffers. I am new to this so do pardon me if I sound stupid!

I have heard of people using tween in PBST and milk in western blot blocking buffers to prevent non-specific binding. However, how does all these blocking substances (tween or milk for example) work? How should one determine their concentration in the whole buffer? And how do we know what other ingredients can be used to prevent non-specific binding or for more stringent washings?

Thanks in advance!



tween will help to wash out proteins that bound non specifically (weak binding). Milk contains protein (you can also use BSA) that will bind via protein protein interactions, non specifically. Then you will have a competition between milk proteins and your antibody with the non specific binding sites. There is a competition for the specific sites, too, but the antibody will win.
As there is an excess of the blocking proteins, you will mainly have milk proteins bound on non specific sites and antibodies on specific sites.
Usually we use up to 5% non fat dry milk or 1% BSA.
You have to try which one is the best, and try different concentrations, increasing if you have background, decreasing if you don't have enough signal.



Dear little mouse,

Thanks for explaining to me.

Other than tween and milk, what other things can we use to wash non-specific binding proteins or used for blocking? it may not necessary be for western blot. I am actually doing an experiment whereby I have streptavidin-coated beads and I have biotinylated anti-HA antibodies bound onto it. At the same time, I have biotinylated templates (with a HA sequence at the 3' end) bound onto the beads too. WIth in vitro translation, proteins (with HA tags at the end) are produced and can bind to the biotinylated anti-HA antibodies on the beads. I am using this peptide conjugated to FITC to detect the proteins, but there is a sign that the peptides are binding nonspecifically to the beads.

The solution would be to have a more stringent washing during the washing steps, or include a blocking buffer during the incubation steps where I am incubating the antibodies and the templates with the beads. Alternatively, I can get a more specific-binding peptide, but that is all I have now and my supervisor would prefer to try out a more stringent washing or a blocking buffer. Is there a website where it carries information on buffers that can be used for such purposes?

Many thanks.

#4 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 03 June 2009 - 05:27 PM

i am sorry to add on. in addition, when do we know when the buffers should be filtered-sterilized?

#5 hanming86

hanming86

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 156 posts
2
Neutral

Posted 03 June 2009 - 07:47 PM

Should it be filter sterilized ? the washing buffer and all that stuff , i made them up and don't even bother sterilizing.
Lab + Coffee + Music = Bliss

#6 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 03 June 2009 - 10:42 PM

Should it be filter sterilized ? the washing buffer and all that stuff , i made them up and don't even bother sterilizing.


Okay thanks a lot!

#7 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 04 June 2009 - 01:16 AM

you can increase the NaCl concentration up to 0.5M to reduce the binding of non biotinylated proteins. Some time it helps.

#8 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 04 June 2009 - 01:17 AM

Other than tween and milk, what other things can we use to wash non-specific binding proteins or used for blocking? it may not necessary be for western blot. I am actually doing an experiment whereby I have streptavidin-coated beads and I have biotinylated anti-HA antibodies bound onto it. At the same time, I have biotinylated templates (with a HA sequence at the 3' end) bound onto the beads too. WIth in vitro translation, proteins (with HA tags at the end) are produced and can bind to the biotinylated anti-HA antibodies on the beads. I am using this peptide conjugated to FITC to detect the proteins, but there is a sign that the peptides are binding nonspecifically to the beads.

The solution would be to have a more stringent washing during the washing steps, or include a blocking buffer during the incubation steps where I am incubating the antibodies and the templates with the beads. Alternatively, I can get a more specific-binding peptide, but that is all I have now and my supervisor would prefer to try out a more stringent washing or a blocking buffer. Is there a website where it carries information on buffers that can be used for such purposes?

Many thanks.



I would block with BSA, not milk, and I would increase the concentration of NaCl up to 0.5M during the washing step.

#9 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 04 June 2009 - 01:37 AM

Hi little mouse,

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.

#10 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 04 June 2009 - 02:18 AM

Hi little mouse,

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.

#11 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 04 June 2009 - 05:06 PM

Hi little mouse,

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.

#12 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 04 June 2009 - 05:15 PM

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.

#13 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 05 June 2009 - 12:23 AM

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

#14 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 05 June 2009 - 12:25 AM

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


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.

#15 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 05 June 2009 - 03:13 AM

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


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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.