I extract protein form tissue sample following AllPrep DNA/RNA/Protein Mini Kit (Qiagen). I dissolve the protein pellet with 100ul SDS 5%. Then I boil at 95°C for 5 min and after that I centrifuge the pellet to collect any residual insoluble material. Then I store @ -20°C my sample till I'm ready to use it for western blot. My question is, should I reboil then samples again before I load them into a gel? Could the samples be damaged by boiling twice?
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.
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)..
Hi, Federica, answer to your question is dependent from your protein properties. But I think that double boiling should not affect your WB results. Below you can find some useful link.
http://www.molecularstation.com/forum/western-blot-forum/74623-how-many-times-do-you-boil-your-western-blot-protein-sample.html
http://biology.stackexchange.com/questions/8404/how-difficult-is-to-renature-a-protein
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
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.
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.
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 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.
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