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查看更多产品信息 Novex™ Reversible Membrane Protein Stain Kit - FAQs (IB7710)
24 个常见问题解答
不能。我们不建议使用胶体蓝（G-250）染色试剂盒对膜进行染色，因为这会产生很高的背景。更好的染色方法包括：
1.Invitrogen可逆性膜蛋白质染料（货号IB7710）：可对硝化纤维素膜和PVDF膜上的蛋白质进行完整的可逆性染色。该产品的灵敏度高于丽春红S（在10分钟内检测到蓝色条带上<10 ng的BSA），可在5分钟内实现可逆性染色。免疫印迹检测不受染色和清除染料过程的影响，在某些情况下可达到更高的灵敏度。
2.Coomassie（非胶体）染色：使用溶于50%甲醇的0.1% Coomassie Blue R-250染色5分钟，然后用50%甲醇和10%乙酸洗涤多次进行脱色。用水洗涤多次、风干并在-20℃下保存，最长可保存12个月。灵敏度约为50-100 ng。
3.SimplyBlue SafeStain染液。SimplyBlue SafeStain使用手册中有对干燥PVDF膜进行染色的实验方案，但不推荐将其用于湿润的PVDF膜或硝化纤维素膜，因为会产生较高的背景。
4.酰胺黑染液：与Coomassie相同，但灵敏度更低。请参见使用手册中的实验方案。
5.丽春红S染液：与Coomassie相同，但灵敏度更低。请参见使用手册中的实验方案。
6.紫外透照法：转印后，将膜放在滤纸上，在室温下干燥10分钟。在20%甲醇中润湿，在湿润状态下于白光前观察印迹；条带会比膜看起来更透明。如果条带随膜变干而消失，则再次润湿膜。
我们建议完成清除步骤，然后再次进行膜染色，并继续完成余下的实验操作。
这可能是因为存在去污剂。染色时，应使用干净的托盘。下游处理，如免疫检测，应单独使用一个托盘。
以下是可能原因和解决方案：
•在膜的某一部位，蛋白浓度较高。降低转印凝胶中的蛋白浓度。
•在某些情况下，经DTT还原的蛋白质可能无法完全清除。可将Eraser溶液的孵育时间延长至5分钟。
使用Eraser溶液可完全清除染料，因此不会影响免疫检测过程。
我们不推荐重复利用Invitrogen可逆性膜蛋白质染色溶液，因为这会导致沉淀形成并影响染色。
与预染ladder中蛋白质结合的染料具有电荷并且是共价结合的，因此，预染ladder的转印效率几乎总是高于SDS变性蛋白。所以，预染ladder不能用于衡量转印成功。
Invitrogen可逆性膜蛋白质染料比丽春红S染料更灵敏。丽春红S不能很好地检测低丰度蛋白质转印或分离的分辨率。
Invitrogen可逆性膜蛋白质染色试剂盒中溶液的成分是保密的。
该染料可检测到<10 ng的牛血清白蛋白。
Invitrogen可逆性膜蛋白质染色试剂盒建议室温保存，可在室温下自收货之日起稳定保存1年。
It is not recommended because the background will be too high. Better alternatives include:
1) Invitrogen Reversible Membrane Protein Stain Kit (Cat. No. IB7710).
2) Coomassie (non-colloidal) staining: stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 min and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at -20°C. Sensitivity is approximately at the 50-100 ng level.
3) Use SimplyBlue SafeStain (Cat. No. LC6060). The SimplyBlue SafeStain manual has the protocol for staining PVDF membranes, but it is not recommended for nitrocellulose because of high background.
4) Amido Black: same as Coomassie but less sensitive.
5) Ponceau S: same as Coomassie but less sensitive.
6) UV transillumination: place membrane on filter paper after blot is finished and allow to dry at room temperature for about 10 min. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear as the membranes dries, rewet again.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Membranes cannot be stained with Colloidal Blue, the background will be too high. Better alternatives include:
1) Coomassie (non-colloidal) staining: Stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 min and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at -20°C. Sensitivity is approximately at the 50-100 ng level.
2) SimplyBlue SafeStain. There is a protocol included in the SimplyBlue manual for staining PVDF but it is not recommended for nitrocellulose because of the high background.
3) Amido Black: less sensitive than Coomassie Blue.
Recipe for amido black: (1L) 450 mL methanol, 450 mL dH2O, 100 mL glacial acetic acid, 0.1 g amido black.
Procedure: combine ingredients and stir until the amido black is dissolved. If the membrane has dried up, pre-wet by floating on dH2O and soaking for 5 min. Transfer to tray containing amido black for 5-10 min. Wash in several changes of dH2O.
4) Ponceau S: less sensitive than Coomassie Blue.
Recipe for Ponceau S (10X stock): 2 g Ponceau S, 30 g trichloroacetic acid, 30 g sulfosalicylic Acid, dH2O to 100 mL
Combine ingredients and stir until Ponceau S is dissolved. Dilute 1:10 before using.
Procedure: If membrane has dried up, pre-wet membrane by floating on dH2O and soaking for 5 min. Transfer membrane to tray containing Ponceau S and incubate for 5-10 min. Wash membrane in several changes of dH2O.
5) UV transillumination: Place membrane on filter paper after blot is finished and allow to dry at room temperature for about 10 min. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear because they dry, rewet again.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
No we do not recommend staining membranes with the Colloidal Blue (G-250) Staining Kit as the background will be too high. Better alternatives include:
- Invitrogen Reversible Membrane Protein Stain (Cat. No. IB7710): Allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. Sensitivity is higher than Ponceau S (<10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes. Western blot detection is unaffected by the staining and erasing process, and in some cases higher sensitivity is achieved.
- Coomassie (non-colloidal) staining: Stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 minutes and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at −20 degrees C. Sensitivity is approximately at the 50-100 ng level.
- SimplyBlue SafeStain. There is a protocol included in the SimplyBlue SafeStain manual for staining dry PVDF membranes but it is not recommended for wet PVDF membranes or nitrocellulose because of the high background.
- Amido black: same as Coomassie stain but less sensitive. Please see protocol in the manual (https://tools.thermofisher.com/content/sfs/manuals/invitrolonpvdf_man.pdf).
- Ponceau S: same as Coomassie stain but less sensitive. Please see protocol in the manual (https://tools.thermofisher.com/content/sfs/manuals/invitrolonpvdf_man.pdf).
- UV transillumination: After blotting, place the membrane on a filter paper and allow to air-dry at room temperature for about 10 minutes. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear because the membrane is dry, rewet the membrane.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We recommend completing the erasure step and then re-staining the membrane and proceeding with the protocol.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This could be due to the presence of detergent. Use clean trays when applying the stain. Use separate trays for downstream processing such as immunodetection.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are possible causes and solutions:
- High protein concentration on specific membrane site. Reduce protein concentration in the transferred gel.
- In some cases, proteins reduced with DTT may not erase completely. Extend erasing step in the Eraser solution for up to 5 minutes.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The stain is completely reversible by using the Eraser solution and hence does not affect the immunodetection process.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We do not recommend re-using the Invitrogen Reversible Membrane Protein Stain solutions as this can lead to formation of a precipitate and will affect the staining.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The dye that is bound to the proteins in the pre-stained ladder is charged and covalently bound, so the transfer efficiency of pre-stained ladders is almost always better than that of SDS-denatured proteins. Therefore, pre-stained ladders are not a good measure for transfer success.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Invitrogen Reversible Membrane Protein Stain is more sensitive than ponceau S. Ponceau S does not give a good measure of low-abundance protein transfer, or of the resolution of the separation.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The composition of the solutions in the Invitrogen Reversible Membrane Protein Stain Kit is proprietary.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The stain is able to detect less than 10 ng bovine serum albumin.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We recommend storing the Invitrogen Reversible Membrane Protein Stain Kit at room temperature where it is stable for one year from date of shipment.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.