If you want to eliminate protein-based viscosity, you can eliminate mostly Mucins through acidification of the sample with TFA (50:50), 1% or lower should be OK, and then centrifugation. Mucins and other big complexes will precipitate and the acidified saliva solution will be liquid and less viscous. Note that you will lose part of the proteome, so keep the precipitate (it can be dissolved in basic solvents). I have personally made SPE with the acidic fractions.
"After collection, salivary samples were immediately mixed with an equal volume of 0.2% aqueous trifluoroacetic acid (TFA) (v/v) in ice bath. After stirring, the acidic supernatant was immediately centrifuged at 9000 × g for 3 min to remove precipitate and the acidic clear solution was either immediately analyzed by HPLC-ESI-MS (100 μl, corresponding to 50 μl of whole saliva) or stored at −80 °C."
If you want to eliminate protein-based viscosity, you can eliminate mostly Mucins through acidification of the sample with TFA (50:50), 1% or lower should be OK, and then centrifugation. Mucins and other big complexes will precipitate and the acidified saliva solution will be liquid and less viscous. Note that you will lose part of the proteome, so keep the precipitate (it can be dissolved in basic solvents). I have personally made SPE with the acidic fractions.
"After collection, salivary samples were immediately mixed with an equal volume of 0.2% aqueous trifluoroacetic acid (TFA) (v/v) in ice bath. After stirring, the acidic supernatant was immediately centrifuged at 9000 × g for 3 min to remove precipitate and the acidic clear solution was either immediately analyzed by HPLC-ESI-MS (100 μl, corresponding to 50 μl of whole saliva) or stored at −80 °C."
The increase in viscosity is usually caused by a protein reaction with air (oxygen). Try pretreating your samples with a solution of the enzyme Trypsin.
The increase in viscosity is usually caused by a protein reaction with air (oxygen). Try pretreating your samples with a solution of the enzyme Trypsin.
Dear Mohammad, Normally we do dilute and shut, the sensitivity of the LC/MS-MS will do it, as you reduce the matrix by dilution. try 1/10. I know that it sounds not logic but try it. best regards
Dear Mohammad, Normally we do dilute and shut, the sensitivity of the LC/MS-MS will do it, as you reduce the matrix by dilution. try 1/10. I know that it sounds not logic but try it. best regards
Thanks Dear Eleuterio Umpierrez i collect saliva by Quantisal device, and extract it by microdispersive extraction, i am using ACN as a dipersive solvent and chloroform as a extracting solvent. but when i collect saliva by quantisal swab and introduce the swab in quantisal liquid. at the end of extraction i have just a hard gel like structure in the bottom of my tube. i used ACN, ACN+2% acetic acid, ethyl acetate and cyclohexane but sitll i coud not to decrease of block gel formation
Thanks Dear Eleuterio Umpierrez i collect saliva by Quantisal device, and extract it by microdispersive extraction, i am using ACN as a dipersive solvent and chloroform as a extracting solvent. but when i collect saliva by quantisal swab and introduce the swab in quantisal liquid. at the end of extraction i have just a hard gel like structure in the bottom of my tube. i used ACN, ACN+2% acetic acid, ethyl acetate and cyclohexane but sitll i coud not to decrease of block gel formation
The gel that you area seeing is the quantisal gel. (ACN or chloroform are doing it) Normally you have the drugs of interest in the buffer, you take it out and do the extraction method that you want (SPE or L/L). If you want it more sensitivity you could add for instants NH4OH to change the pH for specific drugs.
The gel that you area seeing is the quantisal gel. (ACN or chloroform are doing it) Normally you have the drugs of interest in the buffer, you take it out and do the extraction method that you want (SPE or L/L). If you want it more sensitivity you could add for instants NH4OH to change the pH for specific drugs.
If you try a solvent, try Methanol (it might poison the at fault protein).
If you try a solvent, try Methanol (it might poison the at fault protein).
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VOTE
If you want to eliminate protein-based viscosity, you can eliminate mostly Mucins through acidification of the sample with TFA (50:50), 1% or lower should be OK, and then centrifugation. Mucins and other big complexes will precipitate and the acidified saliva solution will be liquid and less viscous. Note that you will lose part of the proteome, so keep the precipitate (it can be dissolved in basic solvents).
I have personally made SPE with the acidic fractions.
"After collection, salivary samples were immediately mixed with an equal volume of 0.2% aqueous trifluoroacetic acid (TFA) (v/v) in ice bath. After stirring, the acidic supernatant was immediately centrifuged at 9000 × g for 3 min to remove precipitate and the acidic clear solution was either immediately analyzed by HPLC-ESI-MS (100 μl, corresponding to 50 μl of whole saliva) or stored at −80 °C."
from:
Molecular & Cellular Proteomics January 1, 2011, First published on October 13, 2010, 10 (1) M110.003467; https://doi.org/10.1074/mcp.M110.003467
If you want to eliminate protein-based viscosity, you can eliminate mostly Mucins through acidification of the sample with TFA (50:50), 1% or lower should be OK, and then centrifugation. Mucins and other big complexes will precipitate and the acidified saliva solution will be liquid and less viscous. Note that you will lose part of the proteome, so keep the precipitate (it can be dissolved in basic solvents).
I have personally made SPE with the acidic fractions.
"After collection, salivary samples were immediately mixed with an equal volume of 0.2% aqueous trifluoroacetic acid (TFA) (v/v) in ice bath. After stirring, the acidic supernatant was immediately centrifuged at 9000 × g for 3 min to remove precipitate and the acidic clear solution was either immediately analyzed by HPLC-ESI-MS (100 μl, corresponding to 50 μl of whole saliva) or stored at −80 °C."
from:
Molecular & Cellular Proteomics January 1, 2011, First published on October 13, 2010, 10 (1) M110.003467; https://doi.org/10.1074/mcp.M110.003467
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VOTE
Thanks dear Roberto Bio
For sure i will try it.
Thanks dear Roberto Bio
For sure i will try it.
More
VOTE
The increase in viscosity is usually caused by a protein reaction with air (oxygen). Try pretreating your samples with a solution of the enzyme Trypsin.
The increase in viscosity is usually caused by a protein reaction with air (oxygen). Try pretreating your samples with a solution of the enzyme Trypsin.
More
VOTE
Dear Mohammad,
Normally we do dilute and shut, the sensitivity of the LC/MS-MS will do it, as you reduce the matrix by dilution. try 1/10. I know that it sounds not logic but try it.
best regards
Dear Mohammad,
Normally we do dilute and shut, the sensitivity of the LC/MS-MS will do it, as you reduce the matrix by dilution. try 1/10. I know that it sounds not logic but try it.
best regards
More
VOTE
Thanks Dear Eleuterio Umpierrez
i collect saliva by Quantisal device, and extract it by microdispersive extraction, i am using ACN as a dipersive solvent and chloroform as a extracting solvent. but when i collect saliva by quantisal swab and introduce the swab in quantisal liquid. at the end of extraction i have just a hard gel like structure in the bottom of my tube. i used ACN, ACN+2% acetic acid, ethyl acetate and cyclohexane but sitll i coud not to decrease of block gel formation
Thanks Dear Eleuterio Umpierrez
i collect saliva by Quantisal device, and extract it by microdispersive extraction, i am using ACN as a dipersive solvent and chloroform as a extracting solvent. but when i collect saliva by quantisal swab and introduce the swab in quantisal liquid. at the end of extraction i have just a hard gel like structure in the bottom of my tube. i used ACN, ACN+2% acetic acid, ethyl acetate and cyclohexane but sitll i coud not to decrease of block gel formation
More
VOTE
The gel that you area seeing is the quantisal gel. (ACN or chloroform are doing it)
Normally you have the drugs of interest in the buffer, you take it out and do the extraction method that you want (SPE or L/L).
If you want it more sensitivity you could add for instants NH4OH to change the pH for specific drugs.
The gel that you area seeing is the quantisal gel. (ACN or chloroform are doing it)
Normally you have the drugs of interest in the buffer, you take it out and do the extraction method that you want (SPE or L/L).
If you want it more sensitivity you could add for instants NH4OH to change the pH for specific drugs.
More
VOTE
Thanks Bruce Neagle,
i well try to solve this problem by a solvent, if i fail, then for sure enzyme is th last choice.
Thanks Bruce Neagle,
i well try to solve this problem by a solvent, if i fail, then for sure enzyme is th last choice.
More
VOTE