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Is it possible to boil protein twice before using it in Western-Blot?
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+ Sds-page
+ Protein denaturation
+ Western blot
+ Protein detection
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Julia Ames
Is it possible to boil protein twice before using it in Western-Blot?
Hello Federica, I have tried both ways ( once and twice boiling), got no significant difference in the W.B results. Therefore, I would say that you do not need to boil your protein twice. Good LUCK Ahmed
Hello Federica, I have tried both ways ( once and twice boiling), got no significant difference in the W.B results. Therefore, I would say that you do not need to boil your protein twice. Good LUCK Ahmed
I always reboil befor loading the sample onto the gel for 1-2 min after storage at -20°C. But in general I pefer to use the sample right away, without freezing. If you have just 1 day between sample preparation and loading, store it at 4°C or on ice.
I always reboil befor loading the sample onto the gel for 1-2 min after storage at -20°C. But in general I pefer to use the sample right away, without freezing. If you have just 1 day between sample preparation and loading, store it at 4°C or on ice.
If you've boiled your sample once, after freezing you can place the tubes 5 min at 60-80 degrees to make sure the total dissolution of the SDS that precipitates in cold. This is enough. In general and depending on each protein, it is not necessary to boil the sample. In some cases boiling (even one only once) worsens sample resolution. Heating the sampler at 90 ° for 5 min once is effective. It should be tested for each protein.
If you've boiled your sample once, after freezing you can place the tubes 5 min at 60-80 degrees to make sure the total dissolution of the SDS that precipitates in cold. This is enough. In general and depending on each protein, it is not necessary to boil the sample. In some cases boiling (even one only once) worsens sample resolution. Heating the sampler at 90 ° for 5 min once is effective. It should be tested for each protein.
If you've boiled your sample once, after freezing you can place the tubes 5 min at 60-80 degrees to make sure the total dissolution of the SDS that precipitates in cold. This is enough. In general and depending on each protein, it is not necessary to boil the sample. In some cases boiling (even one only once) worsens sample resolution. Heating the sampler at 90 ° for 5 min once is effective. It should be tested for each protein.
If you've boiled your sample once, after freezing you can place the tubes 5 min at 60-80 degrees to make sure the total dissolution of the SDS that precipitates in cold. This is enough. In general and depending on each protein, it is not necessary to boil the sample. In some cases boiling (even one only once) worsens sample resolution. Heating the sampler at 90 ° for 5 min once is effective. It should be tested for each protein.
it is not necessary to boil the sample again. after removing from -20 or -80 , samples should be kept for some time at room temperature so that the SDS dissolved properly and there should be no visible precipitate. For this you can keep your samples at 40/50 c .
it is not necessary to boil the sample again. after removing from -20 or -80 , samples should be kept for some time at room temperature so that the SDS dissolved properly and there should be no visible precipitate. For this you can keep your samples at 40/50 c .
I personally do not re-boil the sample again if it is already in the loading buffer... One aim of the boiling is to denature the proteins, but whether refreezing the sample at -20 °C could reverse the boiling step needs a clarification! However, I have tried this procedure without re-boiling the sample (twice)..
I personally do not re-boil the sample again if it is already in the loading buffer... One aim of the boiling is to denature the proteins, but whether refreezing the sample at -20 °C could reverse the boiling step needs a clarification! However, I have tried this procedure without re-boiling the sample (twice)..
I personally don't want my protein samples getting boiled twice. Avoid it. and see if you can add beta mercaptoethanol (2-Mercaptoethanol) in the dissolving solution you are using, then there will be no need to reboil protein.
I personally don't want my protein samples getting boiled twice. Avoid it. and see if you can add beta mercaptoethanol (2-Mercaptoethanol) in the dissolving solution you are using, then there will be no need to reboil protein.
Thanks Chetanchandra S Josh. I have been try to add 2-Mercaptoethanol to a specif buffer of my kit but I have got problem in protein dissolution, so I decided to use SDS.
Thanks Chetanchandra S Josh. I have been try to add 2-Mercaptoethanol to a specif buffer of my kit but I have got problem in protein dissolution, so I decided to use SDS.
I am having same question. The problem is there is no reducing agent in the storage solution. It's just SDS. With first boling, denaturation takes place. But we also need chemical reduction of protein (if there are disulphide bridges in the protein). I think we need to boil the sample with b mercaptoethanol before we load it on the gel.
I am having same question. The problem is there is no reducing agent in the storage solution. It's just SDS. With first boling, denaturation takes place. But we also need chemical reduction of protein (if there are disulphide bridges in the protein). I think we need to boil the sample with b mercaptoethanol before we load it on the gel.
Hello Federica,
I have tried both ways ( once and twice boiling), got no significant difference in the W.B results. Therefore, I would say that you do not need to boil your protein twice.
Good LUCK
Ahmed
Hello Federica,
I have tried both ways ( once and twice boiling), got no significant difference in the W.B results. Therefore, I would say that you do not need to boil your protein twice.
Good LUCK
Ahmed
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Thanks Ahmed for your answer!
Thanks Ahmed for your answer!
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I always reboil befor loading the sample onto the gel for 1-2 min after storage at -20°C. But in general I pefer to use the sample right away, without freezing. If you have just 1 day between sample preparation and loading, store it at 4°C or on ice.
I always reboil befor loading the sample onto the gel for 1-2 min after storage at -20°C. But in general I pefer to use the sample right away, without freezing. If you have just 1 day between sample preparation and loading, store it at 4°C or on ice.
More
VOTE
If you've boiled your sample once, after freezing you can place the tubes 5 min at 60-80 degrees to make sure the total dissolution of the SDS that precipitates in cold. This is enough.
In general and depending on each protein, it is not necessary to boil the sample. In some cases boiling (even one only once) worsens sample resolution. Heating the sampler at 90 ° for 5 min once is effective. It should be tested for each protein.
If you've boiled your sample once, after freezing you can place the tubes 5 min at 60-80 degrees to make sure the total dissolution of the SDS that precipitates in cold. This is enough.
In general and depending on each protein, it is not necessary to boil the sample. In some cases boiling (even one only once) worsens sample resolution. Heating the sampler at 90 ° for 5 min once is effective. It should be tested for each protein.
More
VOTE
If you've boiled your sample once, after freezing you can place the tubes 5 min at 60-80 degrees to make sure the total dissolution of the SDS that precipitates in cold. This is enough.
In general and depending on each protein, it is not necessary to boil the sample. In some cases boiling (even one only once) worsens sample resolution. Heating the sampler at 90 ° for 5 min once is effective. It should be tested for each protein.
If you've boiled your sample once, after freezing you can place the tubes 5 min at 60-80 degrees to make sure the total dissolution of the SDS that precipitates in cold. This is enough.
In general and depending on each protein, it is not necessary to boil the sample. In some cases boiling (even one only once) worsens sample resolution. Heating the sampler at 90 ° for 5 min once is effective. It should be tested for each protein.
More
VOTE
it is not necessary to boil the sample again. after removing from -20 or -80 , samples should be kept for some time at room temperature so that the SDS dissolved properly and there should be no visible precipitate. For this you can keep your samples at 40/50 c .
it is not necessary to boil the sample again. after removing from -20 or -80 , samples should be kept for some time at room temperature so that the SDS dissolved properly and there should be no visible precipitate. For this you can keep your samples at 40/50 c .
More
VOTE
I personally do not re-boil the sample again if it is already in the loading buffer... One aim of the boiling is to denature the proteins, but whether refreezing the sample at -20 °C could reverse the boiling step needs a clarification! However, I have tried this procedure without re-boiling the sample (twice)..
I personally do not re-boil the sample again if it is already in the loading buffer... One aim of the boiling is to denature the proteins, but whether refreezing the sample at -20 °C could reverse the boiling step needs a clarification! However, I have tried this procedure without re-boiling the sample (twice)..
More
VOTE
Reboiling is not necessary. Just thaw your samples and apply them onto your gel.
Reboiling is not necessary. Just thaw your samples and apply them onto your gel.
More
VOTE
I personally don't want my protein samples getting boiled twice. Avoid it. and see if you can add beta mercaptoethanol (2-Mercaptoethanol) in the dissolving solution you are using, then there will be no need to reboil protein.
I personally don't want my protein samples getting boiled twice. Avoid it. and see if you can add beta mercaptoethanol (2-Mercaptoethanol) in the dissolving solution you are using, then there will be no need to reboil protein.
More
VOTE
Thanks Chetanchandra S Josh. I have been try to add 2-Mercaptoethanol to a specif buffer of my kit but I have got problem in protein dissolution, so I decided to use SDS.
Thanks Chetanchandra S Josh. I have been try to add 2-Mercaptoethanol to a specif buffer of my kit but I have got problem in protein dissolution, so I decided to use SDS.
More
VOTE
I dont see the need for reboiling the sample, just thaw and vortex perhaps a step of centrifugation, there should be no problem
I dont see the need for reboiling the sample, just thaw and vortex perhaps a step of centrifugation, there should be no problem
More
VOTE
I am having same question. The problem is there is no reducing agent in the storage solution. It's just SDS. With first boling, denaturation takes place. But we also need chemical reduction of protein (if there are disulphide bridges in the protein). I think we need to boil the sample with b mercaptoethanol before we load it on the gel.
I am having same question. The problem is there is no reducing agent in the storage solution. It's just SDS. With first boling, denaturation takes place. But we also need chemical reduction of protein (if there are disulphide bridges in the protein). I think we need to boil the sample with b mercaptoethanol before we load it on the gel.
More
VOTE