Resuspension of the protein precipitate can be troublesome if you used dentaurant reagents such as TCA. Using chaotropes and mild surfactants (ms compatibles, such as DOC or rapigest, PSS, trypsin enhancers etc.) can facilite the solubilization and you may further continue to your workkflow by reducing, alkylating and tryptic digestion as a standart proteomic procedure. There are alternatives for high protein bearing food matrices (e.g, salting out precpitation using ammonium sulfate, organic solvent precipitations such as ACN, cold acetone, sulphric acid/tungstate flocculation based precipitaiton, physical separation of proteins like using ultrafiltration membranes (size exclusion theory), PEI polymer precipitation etc.). Among these techniques TCA is frequently used and most applicable one for global proteome studies whether in biological or food matrices. Keep in mind, at final solution TCA should be at 20% for efficient protein isolation and high rpm centrifugation rates may improve the pelleted amount. Duration at 4C is critical to isoelectric precipitation methods such as TCA or TCA/Aceton application. SPE may be excluded within this protocol and you may change your precipitation reagent from meoh to abovementioned suggestions. You can strip your pelleted proteins by applying consecutive acetone washes. Thereby you can get rid of any contaminants and residual TCA.
Resuspension of the protein precipitate can be troublesome if you used dentaurant reagents such as TCA. Using chaotropes and mild surfactants (ms compatibles, such as DOC or rapigest, PSS, trypsin enhancers etc.) can facilite the solubilization and you may further continue to your workkflow by reducing, alkylating and tryptic digestion as a standart proteomic procedure. There are alternatives for high protein bearing food matrices (e.g, salting out precpitation using ammonium sulfate, organic solvent precipitations such as ACN, cold acetone, sulphric acid/tungstate flocculation based precipitaiton, physical separation of proteins like using ultrafiltration membranes (size exclusion theory), PEI polymer precipitation etc.). Among these techniques TCA is frequently used and most applicable one for global proteome studies whether in biological or food matrices. Keep in mind, at final solution TCA should be at 20% for efficient protein isolation and high rpm centrifugation rates may improve the pelleted amount. Duration at 4C is critical to isoelectric precipitation methods such as TCA or TCA/Aceton application. SPE may be excluded within this protocol and you may change your precipitation reagent from meoh to abovementioned suggestions. You can strip your pelleted proteins by applying consecutive acetone washes. Thereby you can get rid of any contaminants and residual TCA.
As H. Liu has already suggested, SPE would have done the work. I think you can try precipitating it using methanol/acetone prior to SPE. Please let us know if anything works!
As H. Liu has already suggested, SPE would have done the work. I think you can try precipitating it using methanol/acetone prior to SPE. Please let us know if anything works!
@Sarah Miller King : if the SPE eluent used contains significant percentage of methanol or acetonitrile, it's then not surprising not to see any precipitation after adding methanol.
@Sarah Miller King : if the SPE eluent used contains significant percentage of methanol or acetonitrile, it's then not surprising not to see any precipitation after adding methanol.
Did you perform pH adjustment prior to the SPE cleanup step? My experience is that you don't have to cool to get the proteins to denature with methanol, but that it does appear to work significantly better if you are in a slightly acidic environment (QUEChERS, for example). We use methanol to drop out proteins prior to aflatoxin analysis and it works remarkably well.
Normally you would want to drop out the proteins prior to SPE - they have a tendency to come out of solution when you use alcohol as your eluant and can plug your SPE column.
Did you perform pH adjustment prior to the SPE cleanup step? My experience is that you don't have to cool to get the proteins to denature with methanol, but that it does appear to work significantly better if you are in a slightly acidic environment (QUEChERS, for example). We use methanol to drop out proteins prior to aflatoxin analysis and it works remarkably well.
Normally you would want to drop out the proteins prior to SPE - they have a tendency to come out of solution when you use alcohol as your eluant and can plug your SPE column.
@Sarah Miller King , performing protein precipitation before SPE seems to make more sense to me. Have you tried that?
@Sarah Miller King , performing protein precipitation before SPE seems to make more sense to me. Have you tried that?
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Resuspension of the protein precipitate can be troublesome if you used dentaurant reagents such as TCA. Using chaotropes and mild surfactants (ms compatibles, such as DOC or rapigest, PSS, trypsin enhancers etc.) can facilite the solubilization and you may further continue to your workkflow by reducing, alkylating and tryptic digestion as a standart proteomic procedure. There are alternatives for high protein bearing food matrices (e.g, salting out precpitation using ammonium sulfate, organic solvent precipitations such as ACN, cold acetone, sulphric acid/tungstate flocculation based precipitaiton, physical separation of proteins like using ultrafiltration membranes (size exclusion theory), PEI polymer precipitation etc.). Among these techniques TCA is frequently used and most applicable one for global proteome studies whether in biological or food matrices. Keep in mind, at final solution TCA should be at 20% for efficient protein isolation and high rpm centrifugation rates may improve the pelleted amount. Duration at 4C is critical to isoelectric precipitation methods such as TCA or TCA/Aceton application. SPE may be excluded within this protocol and you may change your precipitation reagent from meoh to abovementioned suggestions. You can strip your pelleted proteins by applying consecutive acetone washes. Thereby you can get rid of any contaminants and residual TCA.
Emir
Resuspension of the protein precipitate can be troublesome if you used dentaurant reagents such as TCA. Using chaotropes and mild surfactants (ms compatibles, such as DOC or rapigest, PSS, trypsin enhancers etc.) can facilite the solubilization and you may further continue to your workkflow by reducing, alkylating and tryptic digestion as a standart proteomic procedure. There are alternatives for high protein bearing food matrices (e.g, salting out precpitation using ammonium sulfate, organic solvent precipitations such as ACN, cold acetone, sulphric acid/tungstate flocculation based precipitaiton, physical separation of proteins like using ultrafiltration membranes (size exclusion theory), PEI polymer precipitation etc.). Among these techniques TCA is frequently used and most applicable one for global proteome studies whether in biological or food matrices. Keep in mind, at final solution TCA should be at 20% for efficient protein isolation and high rpm centrifugation rates may improve the pelleted amount. Duration at 4C is critical to isoelectric precipitation methods such as TCA or TCA/Aceton application. SPE may be excluded within this protocol and you may change your precipitation reagent from meoh to abovementioned suggestions. You can strip your pelleted proteins by applying consecutive acetone washes. Thereby you can get rid of any contaminants and residual TCA.
Emir
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Hi Sarah Miller King ,
As H. Liu has already suggested, SPE would have done the work. I think you can try precipitating it using methanol/acetone prior to SPE.
Please let us know if anything works!
Thank you.
Varsha
Hi Sarah Miller King ,
As H. Liu has already suggested, SPE would have done the work. I think you can try precipitating it using methanol/acetone prior to SPE.
Please let us know if anything works!
Thank you.
Varsha
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@Sarah Miller King : if the SPE eluent used contains significant percentage of methanol or acetonitrile, it's then not surprising not to see any precipitation after adding methanol.
@Sarah Miller King : if the SPE eluent used contains significant percentage of methanol or acetonitrile, it's then not surprising not to see any precipitation after adding methanol.
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i highly recommend probe sonication to coax proteins back up into solubility
i highly recommend probe sonication to coax proteins back up into solubility
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Did you perform pH adjustment prior to the SPE cleanup step? My experience is that you don't have to cool to get the proteins to denature with methanol, but that it does appear to work significantly better if you are in a slightly acidic environment (QUEChERS, for example). We use methanol to drop out proteins prior to aflatoxin analysis and it works remarkably well.
Normally you would want to drop out the proteins prior to SPE - they have a tendency to come out of solution when you use alcohol as your eluant and can plug your SPE column.
Did you perform pH adjustment prior to the SPE cleanup step? My experience is that you don't have to cool to get the proteins to denature with methanol, but that it does appear to work significantly better if you are in a slightly acidic environment (QUEChERS, for example). We use methanol to drop out proteins prior to aflatoxin analysis and it works remarkably well.
Normally you would want to drop out the proteins prior to SPE - they have a tendency to come out of solution when you use alcohol as your eluant and can plug your SPE column.
More
VOTE