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
- - - - -

Newbie question: how to determine protein concentration after IP?


  • Please log in to reply
8 replies to this topic

#1 dc984

dc984

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 05 August 2009 - 03:25 AM

Hello everyone, I'm new here. I have a newbie question.

I am purifying protein lysates by IP, for Western blotting. In the final IP step, it says to elute my antigen from the beads using loading buffer.

Question 1: I need to quantify the protein concentration after IP, to load wells evenly, right??? But I never see people doing that in research papers...is it so simple that it's implied, or is there some other way of going around it?

Question 2: Can I measure protein concentration using Bio-rad protein assay after adding loading buffer? I'm guessing maybe not, because the loading buffer contains dye and other stuff.

Thanks very much!!

#2 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 05 August 2009 - 03:49 AM

not so easy to quantify protein concentration in loading buffer (even if you omit the bromophenol blue). Usually I load all the sample and consider it is quite equally loaded. I was analysing the phospohorylation state of the IPed protein (it was before they sell all this antibodies directed against the phosphorylated protein of interest) with anti-phospho antibodies that detect any phosphorylated protein, stripping and labeling the protein with a normal anti-protein to show that it was equally loaded.
It depend what you want to show.

#3 Dr Teeth

Dr Teeth

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 221 posts
1
Neutral

Posted 05 August 2009 - 04:28 AM

You don't see people doing it because it doesn't make sense to measure protein concentration after IP. Different antibodies will pull down different proteins from different complexes in different amounts, so there is no such thing as an "equally loaded" amount. The best strategy is to load all of your elutant in a single well. You can stain for actin, which should be present in your inputs but absent in your IPs (unless your protein target associates with actin). But, as usual, the best way to be sure of your results is through repeated experimentation.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#4 dc984

dc984

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 05 August 2009 - 05:59 PM

Thanks very much for your replies.

Little mouse, that is what I am trying to measure too, the level of phospho-protein plus level of protein. The question I had was, how to make sure the total (normal) protein would be constant across all wells after IP?

Does this mean I have to treat each sample EXACTLY the same way, so that the volume/concentration across all eluants is the same?? :)

#5 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 05 August 2009 - 11:11 PM

In your case it is very simple, you have an internal control (the level of protein of interest).
Treat your samples exactly the same. the tricky point is the washing step of the beads. You wash 3 times, let always a thin layer of liquid on the top of the beads not to lose beads.
It's only during the last wash that you discard all the liquid, just before to add the loading buffer.
Then you reveal the phosphorylated protein (start by the anti-phospho as the band are weaker than for the total protein), strip and reveal with your normal antibody directed against the protein of interest. Then you can quantify your bands using a software as ImageJ (you will always have slight difference in the total amount), and present the ratio phosphorylated protein versus total protein. To be convincing I was publishing the blot where the total protein was slightly lower when the phosphorylated was clearly higher, so nobody could say there was a bias. (the perfect solution would be to have really equal level)

#6 Penguin

Penguin

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 64 posts
1
Neutral

Posted 06 August 2009 - 04:12 AM

The way I do it is to measure the protein concentration of the cell lysate and then split it evenly between my antibodies. After doing this a few times I've determined that for my purposes 150 ug of cell lysate is optimum so to compare different experiments on different days I always use this amount of lysate.

N.B. If your lysis buffer contains Triton X-100 (as mine does) you cannot use the Bradford assay to quantitate, I use the BCA assay instead.

P

#7 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 06 August 2009 - 04:53 AM

The way I do it is to measure the protein concentration of the cell lysate and then split it evenly between my antibodies. After doing this a few times I've determined that for my purposes 150 ug of cell lysate is optimum so to compare different experiments on different days I always use this amount of lysate.

N.B. If your lysis buffer contains Triton X-100 (as mine does) you cannot use the Bradford assay to quantitate, I use the BCA assay instead.

P


But if you would be sure to load exactly the same amount , this is not enough, because much more variations occur during the immunoprecipitation process than during the cell lysis process. if you don't mesure the concentration right before loading you need an additional control

#8 dc984

dc984

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 07 August 2009 - 06:51 PM

Thanks Penguin and Little mouse for your replies. I am starting to do it now, let's see how it goes!

Little mouse, I have another question :huh:

I've read that you must add enzyme inhibitors fresh to the lysis buffer before use, that I did. I guess it is because the inhibitors lose potency over time.

My question is, if I repeated freeze/thaw my protein lysate (after lysis, before IP), would that have adverse consequences on my sample? Would the inhibitors be degraded, yet the enzymes still active?

The reason is because after obtaining the protein lysis, I freezed the protein samples at -80C and went home. The next day, I thawed them to measure the protein concentration, then freezed them again.

How easily do phosphate groups "fall off"? Should I have somehow deactivated the enzymes first?

Thanks in advance everyone!!! :lol:

#9 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 24 August 2009 - 04:03 AM

Sorry, I was out of the lab.
you have to be careful with crude samples, but when you IP and start purifying, you can stop adding inhibitors, but always keep your samples on ice.
What I do is that I incubate the samples with the antibodies in lysis buffer containing fresh inhibitors, but then I wash with lysis buffer without buffer if don't have buffer containing inhibitors anymore (I have pellets for 10 mL buffer).
about freezing and thawing... I don't think there is a problem. actually I have no answer, but I would not add again inhibitors, I would just keep the sample on ice and quickly bring them back to the freezer. Usually I quantify right after lysis, freeze, and thaw for IP.




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

©1999-2013 Protocol Online, All rights reserved.