如何用SDS-PAGE法去除小分子多肽?

您的樣(yàng)品中是否含有<20 kDa的目标蛋白？您是否發(fā)現常規方法很難分離和檢測小的蛋白和多肽？ 請點擊下載關于 用SDS-PAGE法分離小分子蛋白或者多肽的方法。

Shown is the resolution of cyanogen bromide fragments of myoglobin by (a) Tricine–SDS-PAGE and (b) Laemmli–SDS-PAGE using 10% T, 3% C gels.

Tricine-SDS-PAGE被(bèi)普遍用于分離分子量爲1-100 kDa的蛋白，它被(bèi)認爲是分辨<30 kDa蛋白的首選電泳系統。

請下載Tricine-SDS-PAGE方案，其中包括考瑪斯亮藍染色和電泳的實驗方法。

Tricine-SDS-PAGE is commonly used to separate proteins in the mass range of 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa.

It is indeed very difficult to see the small peptide by SDS-PAGE. Tris-tricine gel will give you a better resolution. If you just want to detect the peptide, Mass Spec is still the best way to confirm the peptide identity.

Small peptide binds less Coomassie brilliant blue than larger protein. Thus smaller peptides are harder to detect by coomassie staining or silver staining. If you really want to see your peptide on the gel, you can try to load more samples. Changing the gel percentage won't help much unless you think your peptide migrated out of the gel. You can increase the percentage of cross linker in the regular 17% gel. In addition increase the pH of your resolving gel to 9.5 as compared to your regular 8.8. Plus, the addition of urea (4-8M) helps sharpen bands.

If you are going to use western, which is a way more sensitive detection method, please use Western instead of the gel staining. However the peptide may simply pass through the membrane. If you repeat the experiment, try to put two pieces of membrane and shorter time of transfer (less than 1 hour at 200 mA). 0.2um pore could be enough. You can get smaller pore but that shouldn't be necessary. You may want to try semi-dry transfer for 15-20 minutes at the recommended current density (mA/cm2) for the apparatus. A short 15 min transfer time works for most of the small peptides.

If you can plan ahead and synthesize a control small peptide labeled with biotin, you can monitor the transfer process and its ability to bind the membrane with streptavidin-conjugated HRP.

Please download this protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue staining, silver staining, and electroblotting.

方案下載

• 請點擊以下按鈕，下載Tricine-SDS-PAGE方案