Home >
Community >
What is an acceptable RZ value for HRP conjugated recombinant...
Upvote
30
Downvote
+ Immunology
+ Protein characterization
+ Horseradish peroxidase
+ Recombinant proteins
Posted by
Alan Waller
What is an acceptable RZ value for HRP conjugated recombinant...
Hi there, I would like to follow up on your previous conversation. Currently, I am facing the problem with the HRP - Ab conjugation ratio.
We have made recombinant 6xHisTag purified HRP, which I need to conjugate with the antibody. The product works fine, but I have a problem with the reproducibility of the process. I am considering, that the possible problem may lie in the incorrect determination of the HRP concentration.
Would you consider being sufficient just to make standard curve of different HRP concentrations at 403nm and simply calculate the concentration of my product? Would it give reliable results for me, or it is necessary to measure activity of the enzyme (e.g. pyrogallol)?
Hi there, I would like to follow up on your previous conversation. Currently, I am facing the problem with the HRP - Ab conjugation ratio.
We have made recombinant 6xHisTag purified HRP, which I need to conjugate with the antibody. The product works fine, but I have a problem with the reproducibility of the process. I am considering, that the possible problem may lie in the incorrect determination of the HRP concentration.
Would you consider being sufficient just to make standard curve of different HRP concentrations at 403nm and simply calculate the concentration of my product? Would it give reliable results for me, or it is necessary to measure activity of the enzyme (e.g. pyrogallol)?
I do not have any publication on this topic at the moment but I am working with phylogenetically related fungal heme peroxidases that also have some fusions and they have a RZ value below 0.9 only when they are not fully saturated with heme.
I do not have any publication on this topic at the moment but I am working with phylogenetically related fungal heme peroxidases that also have some fusions and they have a RZ value below 0.9 only when they are not fully saturated with heme.
The RZ give you some hibt about the purity of the HRP or HRP labelled proteins. The pure HRP has an RZ of 3 ... 3.5. The RZ of conjugates depends on the molar ratio of the conjugation (mol HRP per mol protein to label), the size of ypor protein you are conugating. At al, it's not a good quality parameter to describe the quality of your conjugate. The RZ of a conjugated protein can be helpful to compare different coupling procedures, different lots, ... Why? In immune reactions detected by labelled antigens / antibodies the affinity determines of the antigen/antibody interaction the detection limit. The labeled partner hat to be in excess present in this reaction. Therefore, also the substrate should never be the limiting part in an ELISA.
The RZ give you some hibt about the purity of the HRP or HRP labelled proteins. The pure HRP has an RZ of 3 ... 3.5. The RZ of conjugates depends on the molar ratio of the conjugation (mol HRP per mol protein to label), the size of ypor protein you are conugating. At al, it's not a good quality parameter to describe the quality of your conjugate. The RZ of a conjugated protein can be helpful to compare different coupling procedures, different lots, ... Why? In immune reactions detected by labelled antigens / antibodies the affinity determines of the antigen/antibody interaction the detection limit. The labeled partner hat to be in excess present in this reaction. Therefore, also the substrate should never be the limiting part in an ELISA.
The RZ give you some hibt about the purity of the HRP or HRP labelled proteins. The pure HRP has an RZ of 3 ... 3.5. The RZ of conjugates depends on the molar ratio of the conjugation (mol HRP per mol protein to label), the size of ypor protein you are conugating. At al, it's not a good quality parameter to describe the quality of your conjugate. The RZ of a conjugated protein can be helpful to compare different coupling procedures, different lots, ... Why? In immune reactions detected by labelled antigens / antibodies the affinity determines of the antigen/antibody interaction the detection limit. The labeled partner hat to be in excess present in this reaction. Therefore, also the substrate should never be the limiting part in an ELISA.
The RZ give you some hibt about the purity of the HRP or HRP labelled proteins. The pure HRP has an RZ of 3 ... 3.5. The RZ of conjugates depends on the molar ratio of the conjugation (mol HRP per mol protein to label), the size of ypor protein you are conugating. At al, it's not a good quality parameter to describe the quality of your conjugate. The RZ of a conjugated protein can be helpful to compare different coupling procedures, different lots, ... Why? In immune reactions detected by labelled antigens / antibodies the affinity determines of the antigen/antibody interaction the detection limit. The labeled partner hat to be in excess present in this reaction. Therefore, also the substrate should never be the limiting part in an ELISA.
Dear all, thanks for your answers. yes Marcia, of course I will perform a capture ELISA in order to detect the activity of the conjugates, but I would like to know a little more about the possibility of this parameter, RZ. Thank you Dorotea, I´ll do. Dear Marcel, your answer is very interesting, thank you for the information. Please, let me know if you have knowledge about an interesting publication regarding this parameter.
Dear all, thanks for your answers. yes Marcia, of course I will perform a capture ELISA in order to detect the activity of the conjugates, but I would like to know a little more about the possibility of this parameter, RZ. Thank you Dorotea, I´ll do. Dear Marcel, your answer is very interesting, thank you for the information. Please, let me know if you have knowledge about an interesting publication regarding this parameter.
(just returned from vacations) To Camille Solbrig: for heme peroxidases in general and HRP in particular NO amino acid acts as reactant, H2O2 or peroxidic bond in any molecule (also in organic peroxides!) acts as an electron acceptor to activate HRP or other peroxidase to compound I where it can accept electrons from other substances, particularly florescence dyes that are used in ELISA. To Stephan Kiessig, I am not working with HRP conjugates but with other natural peroxidase conjugates and I think that RZ value is an important hint on the purity of the fused protein. Surely for specific ELISA applications there are other parameters that do matter. But at least in complex experiments the obtained RZ value gives the lucky researcher a control value to be sure that (if not working) from the side of peroxidase it is acceptable or not ...
Biraza Sethani Academy Biraza Sethani AcademyFollowFollowing
(just returned from vacations) To Camille Solbrig: for heme peroxidases in general and HRP in particular NO amino acid acts as reactant, H2O2 or peroxidic bond in any molecule (also in organic peroxides!) acts as an electron acceptor to activate HRP or other peroxidase to compound I where it can accept electrons from other substances, particularly florescence dyes that are used in ELISA. To Stephan Kiessig, I am not working with HRP conjugates but with other natural peroxidase conjugates and I think that RZ value is an important hint on the purity of the fused protein. Surely for specific ELISA applications there are other parameters that do matter. But at least in complex experiments the obtained RZ value gives the lucky researcher a control value to be sure that (if not working) from the side of peroxidase it is acceptable or not ...
I am not familiar with RZ values. However since it deals with absorbtion values it probably measures the amount of protein which is related to the amino acid that causes the absorption. So any test or knowledge of how much of the amino acid that is necessary to act as a reactant in the conjugation reaction will give you the information that you want.
I am not familiar with RZ values. However since it deals with absorbtion values it probably measures the amount of protein which is related to the amino acid that causes the absorption. So any test or knowledge of how much of the amino acid that is necessary to act as a reactant in the conjugation reaction will give you the information that you want.
Your problem is complicated with the presence of a HRP - Ab conjugate. As mentioned above the RZ value is indicative of peroxidase purity. If you have different samples the RZ i.e. the ratio between A403nm and A280nm shall remain constant. If it is falling (in some samples) then you have less heme-saturared peroxidase. But you can salso determine the actual HRP concentraion directly via the A403nm absorption supposing that it is fully saturated with heme ...
Your problem is complicated with the presence of a HRP - Ab conjugate. As mentioned above the RZ value is indicative of peroxidase purity. If you have different samples the RZ i.e. the ratio between A403nm and A280nm shall remain constant. If it is falling (in some samples) then you have less heme-saturared peroxidase. But you can salso determine the actual HRP concentraion directly via the A403nm absorption supposing that it is fully saturated with heme ...
I think a RZ value of 1.0 - 1.5 would be acceptable for a HRP-conjugate. However, the final RZ depends on the total length of the construct indirect proportionally. For shorter conjugates values towards 1.5 would be awaited and for longer constucts down to 1.0 If the measured value is far below 1.0 this could indicate that the peroxidase domain of HRP is not fully saturated with the prosthetic heme group!
I think a RZ value of 1.0 - 1.5 would be acceptable for a HRP-conjugate. However, the final RZ depends on the total length of the construct indirect proportionally. For shorter conjugates values towards 1.5 would be awaited and for longer constucts down to 1.0 If the measured value is far below 1.0 this could indicate that the peroxidase domain of HRP is not fully saturated with the prosthetic heme group!
Dear Ana, I'm so sorry, but I haven't. Nowadays, I'm in the field of food biotechnology and I'm not in enzymes research. Maybe you can ask some of my collegues of the article. best wishes
Dear Ana, I'm so sorry, but I haven't. Nowadays, I'm in the field of food biotechnology and I'm not in enzymes research. Maybe you can ask some of my collegues of the article. best wishes
Hi there, I would like to follow up on your previous conversation. Currently, I am facing the problem with the HRP - Ab conjugation ratio.
We have made recombinant 6xHisTag purified HRP, which I need to conjugate with the antibody. The product works fine, but I have a problem with the reproducibility of the process. I am considering, that the possible problem may lie in the incorrect determination of the HRP concentration.
Would you consider being sufficient just to make standard curve of different HRP concentrations at 403nm and simply calculate the concentration of my product? Would it give reliable results for me, or it is necessary to measure activity of the enzyme (e.g. pyrogallol)?
Hi there, I would like to follow up on your previous conversation. Currently, I am facing the problem with the HRP - Ab conjugation ratio.
We have made recombinant 6xHisTag purified HRP, which I need to conjugate with the antibody. The product works fine, but I have a problem with the reproducibility of the process. I am considering, that the possible problem may lie in the incorrect determination of the HRP concentration.
Would you consider being sufficient just to make standard curve of different HRP concentrations at 403nm and simply calculate the concentration of my product? Would it give reliable results for me, or it is necessary to measure activity of the enzyme (e.g. pyrogallol)?
More
VOTE
I do not have any publication on this topic at the moment but I am working with phylogenetically related fungal heme peroxidases that also have some fusions and they have a RZ value below 0.9 only when they are not fully saturated with heme.
I do not have any publication on this topic at the moment but I am working with phylogenetically related fungal heme peroxidases that also have some fusions and they have a RZ value below 0.9 only when they are not fully saturated with heme.
More
VOTE
The RZ give you some hibt about the purity of the HRP or HRP labelled proteins. The pure HRP has an RZ of 3 ... 3.5. The RZ of conjugates depends on the molar ratio of the conjugation (mol HRP per mol protein to label), the size of ypor protein you are conugating.
At al, it's not a good quality parameter to describe the quality of your conjugate. The RZ of a conjugated protein can be helpful to compare different coupling procedures, different lots, ...
Why? In immune reactions detected by labelled antigens / antibodies the affinity determines of the antigen/antibody interaction the detection limit. The labeled partner hat to be in excess present in this reaction. Therefore, also the substrate should never be the limiting part in an ELISA.
The RZ give you some hibt about the purity of the HRP or HRP labelled proteins. The pure HRP has an RZ of 3 ... 3.5. The RZ of conjugates depends on the molar ratio of the conjugation (mol HRP per mol protein to label), the size of ypor protein you are conugating.
At al, it's not a good quality parameter to describe the quality of your conjugate. The RZ of a conjugated protein can be helpful to compare different coupling procedures, different lots, ...
Why? In immune reactions detected by labelled antigens / antibodies the affinity determines of the antigen/antibody interaction the detection limit. The labeled partner hat to be in excess present in this reaction. Therefore, also the substrate should never be the limiting part in an ELISA.
More
VOTE
The RZ give you some hibt about the purity of the HRP or HRP labelled proteins. The pure HRP has an RZ of 3 ... 3.5. The RZ of conjugates depends on the molar ratio of the conjugation (mol HRP per mol protein to label), the size of ypor protein you are conugating.
At al, it's not a good quality parameter to describe the quality of your conjugate. The RZ of a conjugated protein can be helpful to compare different coupling procedures, different lots, ...
Why? In immune reactions detected by labelled antigens / antibodies the affinity determines of the antigen/antibody interaction the detection limit. The labeled partner hat to be in excess present in this reaction. Therefore, also the substrate should never be the limiting part in an ELISA.
The RZ give you some hibt about the purity of the HRP or HRP labelled proteins. The pure HRP has an RZ of 3 ... 3.5. The RZ of conjugates depends on the molar ratio of the conjugation (mol HRP per mol protein to label), the size of ypor protein you are conugating.
At al, it's not a good quality parameter to describe the quality of your conjugate. The RZ of a conjugated protein can be helpful to compare different coupling procedures, different lots, ...
Why? In immune reactions detected by labelled antigens / antibodies the affinity determines of the antigen/antibody interaction the detection limit. The labeled partner hat to be in excess present in this reaction. Therefore, also the substrate should never be the limiting part in an ELISA.
More
VOTE
Dear all, thanks for your answers.
yes Marcia, of course I will perform a capture ELISA in order to detect the activity of the conjugates, but I would like to know a little more about the possibility of this parameter, RZ.
Thank you Dorotea, I´ll do.
Dear Marcel, your answer is very interesting, thank you for the information. Please, let me know if you have knowledge about an interesting publication regarding this parameter.
Dear all, thanks for your answers.
yes Marcia, of course I will perform a capture ELISA in order to detect the activity of the conjugates, but I would like to know a little more about the possibility of this parameter, RZ.
Thank you Dorotea, I´ll do.
Dear Marcel, your answer is very interesting, thank you for the information. Please, let me know if you have knowledge about an interesting publication regarding this parameter.
More
VOTE
(just returned from vacations) To Camille Solbrig: for heme peroxidases in general and HRP in particular NO amino acid acts as reactant, H2O2 or peroxidic bond in any molecule (also in organic peroxides!) acts as an electron acceptor to activate HRP or other peroxidase to compound I where it can accept electrons from other substances, particularly florescence dyes that are used in ELISA. To Stephan Kiessig, I am not working with HRP conjugates but with other natural peroxidase conjugates and I think that RZ value is an important hint on the purity of the fused protein. Surely for specific ELISA applications there are other parameters that do matter. But at least in complex experiments the obtained RZ value gives the lucky researcher a control value to be sure that (if not working) from the side of peroxidase it is acceptable or not ...
(just returned from vacations) To Camille Solbrig: for heme peroxidases in general and HRP in particular NO amino acid acts as reactant, H2O2 or peroxidic bond in any molecule (also in organic peroxides!) acts as an electron acceptor to activate HRP or other peroxidase to compound I where it can accept electrons from other substances, particularly florescence dyes that are used in ELISA. To Stephan Kiessig, I am not working with HRP conjugates but with other natural peroxidase conjugates and I think that RZ value is an important hint on the purity of the fused protein. Surely for specific ELISA applications there are other parameters that do matter. But at least in complex experiments the obtained RZ value gives the lucky researcher a control value to be sure that (if not working) from the side of peroxidase it is acceptable or not ...
More
VOTE
I am not familiar with RZ values. However since it deals with absorbtion values it probably measures the amount of protein which is related to the amino acid that causes the absorption. So any test or knowledge of how much of the amino acid that is necessary to act as a reactant in the conjugation reaction will give you the information that you want.
I am not familiar with RZ values. However since it deals with absorbtion values it probably measures the amount of protein which is related to the amino acid that causes the absorption. So any test or knowledge of how much of the amino acid that is necessary to act as a reactant in the conjugation reaction will give you the information that you want.
More
VOTE
Thank you Marcel, in any case this is the information I need. Thank you very much.
Thank you Marcel, in any case this is the information I need. Thank you very much.
More
VOTE
Your problem is complicated with the presence of a HRP - Ab conjugate. As mentioned above the RZ value is indicative of peroxidase purity. If you have different samples the RZ i.e. the ratio between A403nm and A280nm shall remain constant. If it is falling (in some samples) then you have less heme-saturared peroxidase. But you can salso determine the actual HRP concentraion directly via the A403nm absorption supposing that it is fully saturated with heme ...
Your problem is complicated with the presence of a HRP - Ab conjugate. As mentioned above the RZ value is indicative of peroxidase purity. If you have different samples the RZ i.e. the ratio between A403nm and A280nm shall remain constant. If it is falling (in some samples) then you have less heme-saturared peroxidase. But you can salso determine the actual HRP concentraion directly via the A403nm absorption supposing that it is fully saturated with heme ...
More
VOTE
I think a RZ value of 1.0 - 1.5 would be acceptable for a HRP-conjugate. However, the final RZ depends on the total length of the construct indirect proportionally. For shorter conjugates values towards 1.5 would be awaited and for longer constucts down to 1.0 If the measured value is far below 1.0 this could indicate that the peroxidase domain of HRP is not fully saturated with the prosthetic heme group!
I think a RZ value of 1.0 - 1.5 would be acceptable for a HRP-conjugate. However, the final RZ depends on the total length of the construct indirect proportionally. For shorter conjugates values towards 1.5 would be awaited and for longer constucts down to 1.0 If the measured value is far below 1.0 this could indicate that the peroxidase domain of HRP is not fully saturated with the prosthetic heme group!
More
VOTE
Dear Ana,
I'm so sorry, but I haven't. Nowadays, I'm in the field of food biotechnology and I'm not in enzymes research. Maybe you can ask some of my collegues of the article.
best wishes
Dear Ana,
I'm so sorry, but I haven't. Nowadays, I'm in the field of food biotechnology and I'm not in enzymes research. Maybe you can ask some of my collegues of the article.
best wishes
More
VOTE