Hi Romain,

AF647 is one of the best dyes for dSTORM, and should blink upon different buffers and reagent concentrations. In my hands I get good blinking from AF647 by using MEA 100mM + Glucose oxidase: I prepare the buffer following the protocol at the bottom.

Regarding the substrate and glass treatment, I would stick to what you normally use for regular IF as it really depends on the cells that you are using. What where the problems with your first dStorm tests?

Best,

Davide

MEA SOLUTION

- Weight 560 mg of Cysteamine HCl

- Dissolve in 30mL of PBS

- Weight 5g of glucose

Add glucose to Cysteamine solution and add PBS up to 50 ml

- Adjust pH to 7.4.

- Aliquot in 2ml tubes and freeze at -20 °C.

Add 1ml of MEA mix to cells just before imaging together with 10µl of 50 mg/ml Glucose Oxidase and 1µl of 40mg/ml catalase.

Hi Davide,

Thanks for the information. But what I am really after is a protocol to prepare in-vitro actin filament on coverslip. It's not so much the imaging part (I have a good experience in dSTORM imaging) but the sample preparation part that is causing trouble.

Do you have experience in preparing actin filament in-vitro?

Thanks anyway for your answer !

Romain

Dear Romain,

Actin from Sigma is usually of poor quality. This means that preps might not polymerise well or contain aggregates. As you only ned to have actin filaments, I would spin G-actin stock solution at 100K x g for 30 min and then induce polymerization. If you just need to image F-actin, let this solution stand ON at RT. When you spot it do not dilute below 0.5 uM. Chop the tip of your pipet thereby avoiding filament fragmentation and add methylcellulose to prevent fast diffusion. As diffusion makes super resolution imaging impossible, you may want to functionalize your coverslips with some actin-binding proteins that will immobilise F-actin. Actin polymerization buffers and protocols are standard.

Good luck,

Metello

Dear Metello,

That's very useful. Many thanks !

Can you send me some links/publications to those standard protocols please?

Thanks again.

ROmain

Dear Metello,

how is it with Actin from cytoskeleton?

Hi Lindave,

Actin from Cytoskeleton is not any better. Thus, after resuspending the powder in G-actin buffer, you should first spin it as I explained in my older post.

Hope this helps.

Cheers,

Metello

 Dear Metello,

A lot of thanks!

Please could you send me some publications/links to those Standard protocols?

"As you only ned to have actin filaments, I would spin G-actin stock solution at 100K x g for 30 min and then induce polymerization. If you just need to image F-actin, let this solution stand ON at RT". I need to image F-actin should I leave the solution at RT after inducing the polymerization or before? and for which time?

Thanks again.

Lindave

Hi Lindave,

You can store G-actin on ice for 3-4 weeks. F-actin can be polymerised at RT. The polymerisation reaction should reach the plateau in 30 min-1 hour (depending on temperature and concentration). For the simple purpose of imaging actin filaments, whether or not F-actin is freshly assembled does not make any difference. This is why in my previous post, I wrote just let it polymerise at RT ON. If you need to carry out biochemical assays, this advice might not be correct. 

Basic protocols about actin:

 Hertzog, M. and Carlier, M. F. (2005). Functional characterization of proteins regulating actin assembly. Curr Protoc Cell Biol Chapter 13, Unit 13 16.

Cheers,

Metello

What exact buffer system should be used to prepare solution of G-Actin from rabbit muscle if I want to use HEPES buffer pH-7.4 ? Also how to make sure that actin doesnot undergo polymerization as well?