SYPRO™ Orange Protein Gel Stain (5000X Concentrate in DMSO), 500 μL - FAQs

不会。染料浓度高于1X不会带来更好的检测结果。相反，随着染料浓度的增加，背景荧光会增强，染料会发生自淬灭，从而降低信号。

经过SYPRO染料染色的凝胶可放置在玻璃纸之间进行干燥，但有时会轻微降低灵敏度。如果在纸上干燥凝胶，光会散射且灵敏度会降低。不推荐使用其他塑料，因为通常使用的塑料是不可透过紫外光的。

可以，您可使用SYPRO Orange蛋白质凝胶染料对Invitrogen凝胶进行染色，但需要更改实验方案。该染料主要是与0.05% SDS电泳缓冲液一起使用，但我们所有的缓冲液均含有0.1% SDS。这会产生较高的背景，因为SDS会与染料紧密结合。以下是获得最佳Invitrogen凝胶染色结果的推荐方案：

洗涤液：7.5% (v/v) 乙酸

染色液：在洗涤液中按1:5000稀释SYPRO Orange
1.凝胶电泳完毕后，在100 ml洗涤液中清洗10分钟。
2.将凝胶置于染色液中，在暗处放置长达24小时。
3.从染色液中取出凝胶，在100 ml洗涤液中短暂清洗。
4.随后，可对凝胶进行拍照。

不能。上样溶液含有很多SDS，因此，SYPRO Ruby、SYPRO Orange和SYPRO Red染料会只与游离的SDS结合，而极少与蛋白质结合。在电泳前，可预先使用ATTO-TAG CBQCA（货号A2333）、DDAO琥珀酰亚胺酯（货号C34553）或TAMRA-琥珀酰亚胺酯（货号C2211）染料或TC-FLAsH表达分析检测试剂盒（货号A10067为橙黄色荧光，货号A10068为红色荧光）对蛋白质进行共价标记，不会影响蛋白质在凝胶中的迁移。

电泳期间，可将SYPRO Orange或SYPRO Red蛋白质凝胶染料稀释5000倍并加入到阴极（上层）缓冲液槽中，对蛋白质进行染色而不影响迁移。这样做可能引起的问题是，染料与SDS的相互作用可能导致凝胶产生背景荧光。在电泳后使用7.5%乙酸脱色15–60分钟，可降低这种背景荧光。这种染色方法还会导致蛋白质灵敏度低于标准的后染色方法、在凝胶成像前需要相同的时间以及污染电泳装置。

No. Dye concentrations higher than 1X do not give better detection. Instead, background fluorescence increases and, as the dye concentration increases, the dye becomes self-quenching and the signal actually decreases.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Gels stained with SYPRO dyes can be dried between sheets of cellophane, although there is sometimes a slight decrease in sensitivity. If the gels are dried onto paper, the light will scatter and the sensitivity will decrease. Other plastics are not recommended, as the plastic typically used is not transparent to UV light.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Yes, you may use SYPRO Orange Protein Gel Stain with Invitrogen gels but the protocol would have to be modified. The stain is mainly to be used with 0.05% SDS running buffer while all our buffers contain 0.1% SDS. This results in high background as the SDS binds tightly to the stain. The following protocol is recommended for best results with Invitrogen gels:

Wash solution: 7.5% (v/v) Acetic acid
Staining solution: 1:5000 SYPRO Orange in Wash solution

After the gel run is over, wash the gel in 100 ml of Wash solution for 10 minutes.
Place gel in Staining solution for up to 24 h in the dark.
Remove the gel from the Staining solution and rinse briefly in 100 ml Wash solution.
The gel is then ready to be photographed.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

No. Loading solutions contain so much SDS that SYPRO Ruby, SYPRO Orange and SYPRO Red dyes simply localize in the free SDS and bind very little of the proteins. Proteins can be covalently pre-labeled with ATTO-TAG CBQCA (Cat. No. A2333), DDAO succinimidyl ester (Cat. No. C34553) or TAMRA-succinimidyl ester (Cat. No. C2211) dyes, or the TC-FLAsH Expression Analysis Detection Kits (Cat. No. A10067 for orange fluorescence, Cat. No. A10068 for red fluorescence) prior to electrophoresis without affecting protein migration through the gel.

SYPRO Orange or SYPRO Red Protein Gel Stain can be diluted 5000-fold into the cathode (upper) buffer tank to stain proteins during electrophoresis without affecting migration. The problem with doing this is that there is considerable background fluorescence in the gels from the dye interacting with SDS. This can be reduced after electrophoresis by destaining in 7.5% acetic acid for 15-60 minutes. This method also results in poorer protein sensitivity than the standard post-staining method, requires the same amount of time before the gel can be imaged, and contaminates the electrophoresis apparatus.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.