Pimp my ELISA plate
January 20, 2012 10:48 AM Subscribe
Calling all protein scientists! When coating an ELISA plate with a target protein, how does the coating buffer pH specifically effect the coating efficiency?
Assume that you're using a high binding polystyrene plate that has been irradiated and a carboxylic acid inserted into the broken benzene rings, and you'll be relying on hydrophobic and electrostatic interactions to drive the adsorption. I would assume that the pH of the coating solution could safely be adjusted through a slightly acidic to a slightly alkaline pH depending on the protein of interest, but I've never seen a plate coated in anything other than neutral to slightly alkaline conditions. In fact, I almost always see the alkaline pH as accepted as best practice.
What gives?
Assume that you're using a high binding polystyrene plate that has been irradiated and a carboxylic acid inserted into the broken benzene rings, and you'll be relying on hydrophobic and electrostatic interactions to drive the adsorption. I would assume that the pH of the coating solution could safely be adjusted through a slightly acidic to a slightly alkaline pH depending on the protein of interest, but I've never seen a plate coated in anything other than neutral to slightly alkaline conditions. In fact, I almost always see the alkaline pH as accepted as best practice.
What gives?
I sort of recall that the alkaline binding buffer is a best-guess approach for when you don't know specifically what conditions are optimal for your target protein, or you're using a mixture like a cell lysate or serum. There are 2 reasons for choosing slightly alkaline pH:
1. It's physiological, more or less, so your proteins are likely to stay undenatured and unprecipitated.
2. Lots of proteins have a fairly high pI so they will be positively charged in a slightly alkaline buffer and will bind to the negatively-charged plate.
Of course, lots of other proteins have a lower pI and won't bind very well, which is another reason why "sandwich" ELISAs became more popular than direct ELISAs - you can optimize binding conditions for your "trap" coating since it will be pretty consistent from batch to batch.
posted by Quietgal at 12:22 PM on January 20, 2012
1. It's physiological, more or less, so your proteins are likely to stay undenatured and unprecipitated.
2. Lots of proteins have a fairly high pI so they will be positively charged in a slightly alkaline buffer and will bind to the negatively-charged plate.
Of course, lots of other proteins have a lower pI and won't bind very well, which is another reason why "sandwich" ELISAs became more popular than direct ELISAs - you can optimize binding conditions for your "trap" coating since it will be pretty consistent from batch to batch.
posted by Quietgal at 12:22 PM on January 20, 2012
Response by poster: Slackermagaee: Just a standard immunospecific ELISA.
posted by Dr. ShadowMask at 9:00 PM on January 20, 2012
posted by Dr. ShadowMask at 9:00 PM on January 20, 2012
A pure polystyrene plate is going to look would be almost uniformly hydrophobic. Have you seen the paper by Matsurra et al. re: the incorporation of oxygen? They report the incorporation of oxygen as being both C=O and C-O moieties, but don't specifically say carboxylic acids or that the benzene rings are broken. Since this probably starts out with the addition of a radical oxygen, I'd guess it ends in something more like an aldehyde (or maybe something like 2,4 cyclohexadieneone) but I haven't gotten out a paper and pencil to play with all the potential resonance structures to figure out which one's the carbon atoms would be happiest with.
Either way, you end up with a mostly hydrophobic plane with periodic negative charges on it (the oxygens). Just playing the odds that's the way to go because out of twenty amino acids, 11 of them are likely to be happy with this arrangement because they either have hydrophobic or positively charges side chains at neutral pH. By making conditions slightly basic, you'll drive things to be slight more negative and, as Quietgal says, create more sites that will be eager to bind with the negatively charged points on the plate. The ideal pH is going to depend on the physical arrangement of the hydrophobic and positive charges on your protein and how dense the oxygens are on the plate.
Really, you shouldn't anthropomorphize molecules and atoms the way I am because THEY HATE IT WHEN YOU DO THAT!
posted by Kid Charlemagne at 11:33 PM on January 20, 2012
Either way, you end up with a mostly hydrophobic plane with periodic negative charges on it (the oxygens). Just playing the odds that's the way to go because out of twenty amino acids, 11 of them are likely to be happy with this arrangement because they either have hydrophobic or positively charges side chains at neutral pH. By making conditions slightly basic, you'll drive things to be slight more negative and, as Quietgal says, create more sites that will be eager to bind with the negatively charged points on the plate. The ideal pH is going to depend on the physical arrangement of the hydrophobic and positive charges on your protein and how dense the oxygens are on the plate.
Really, you shouldn't anthropomorphize molecules and atoms the way I am because THEY HATE IT WHEN YOU DO THAT!
posted by Kid Charlemagne at 11:33 PM on January 20, 2012
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posted by Slackermagee at 11:37 AM on January 20, 2012