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How much protein do you need to load on a dot blot?
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Kelli Shomaker
How much protein do you need to load on a dot blot?
Hi, Do you have enough materials to make a little curve? That's the best way to aswer your question. Generally using western blot technique your able to detect at the range of pg.
Hi, Do you have enough materials to make a little curve? That's the best way to aswer your question. Generally using western blot technique your able to detect at the range of pg.
Hello, Jackson It depends of the characteristics of your protein (purity, antigenicity, etc) and your Dot blot protocol (one detection antibody or doble antibodies, the substrate, etc). As Mr. Martin told you, you can do serial dilutions and try with a pilot assay, including a negative control. In our lab we had detected concentrations of tetanus toxoid as low as 0.1 ug/ml, and even less. Maybe in your case, a concentration around 0,5 ug/ml (application 5-10 ul) should be enough. If you want, you can include the starting material as control. Be sure to keep space between this control and the samples, because when the concentration is to high, usually it produces an enlarged spot. I hope these suggestions help you. Best regards
Hello, Jackson It depends of the characteristics of your protein (purity, antigenicity, etc) and your Dot blot protocol (one detection antibody or doble antibodies, the substrate, etc). As Mr. Martin told you, you can do serial dilutions and try with a pilot assay, including a negative control. In our lab we had detected concentrations of tetanus toxoid as low as 0.1 ug/ml, and even less. Maybe in your case, a concentration around 0,5 ug/ml (application 5-10 ul) should be enough. If you want, you can include the starting material as control. Be sure to keep space between this control and the samples, because when the concentration is to high, usually it produces an enlarged spot. I hope these suggestions help you. Best regards
Hi,
Do you have enough materials to make a little curve? That's the best way to aswer your question.
Generally using western blot technique your able to detect at the range of pg.
Hi,
Do you have enough materials to make a little curve? That's the best way to aswer your question.
Generally using western blot technique your able to detect at the range of pg.
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Hello, Jackson
It depends of the characteristics of your protein (purity, antigenicity, etc) and your Dot blot protocol (one detection antibody or doble antibodies, the substrate, etc). As Mr. Martin told you, you can do serial dilutions and try with a pilot assay, including a negative control. In our lab we had detected concentrations of tetanus toxoid as low as 0.1 ug/ml, and even less. Maybe in your case, a concentration around 0,5 ug/ml (application 5-10 ul) should be enough. If you want, you can include the starting material as control. Be sure to keep space between this control and the samples, because when the concentration is to high, usually it produces an enlarged spot.
I hope these suggestions help you.
Best regards
Hello, Jackson
It depends of the characteristics of your protein (purity, antigenicity, etc) and your Dot blot protocol (one detection antibody or doble antibodies, the substrate, etc). As Mr. Martin told you, you can do serial dilutions and try with a pilot assay, including a negative control. In our lab we had detected concentrations of tetanus toxoid as low as 0.1 ug/ml, and even less. Maybe in your case, a concentration around 0,5 ug/ml (application 5-10 ul) should be enough. If you want, you can include the starting material as control. Be sure to keep space between this control and the samples, because when the concentration is to high, usually it produces an enlarged spot.
I hope these suggestions help you.
Best regards
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Hi Jackson,
You can apply 1 ul of protein per dot. Do titration dilutions for each antibody to optimise later for Westerns analysis.
Best,
Hussein
Hi Jackson,
You can apply 1 ul of protein per dot. Do titration dilutions for each antibody to optimise later for Westerns analysis.
Best,
Hussein
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