Hello,

You need a table-top ultracentrifuge (e.g. Beckman Optima) and a small rotor (e.g. TLA100) with a max tube volume ~300ul. Also, I would guess that there is no need to maintain 37oC during the spin. I can ask people around to find out the protocol we used to separate the F- and G-actin pools, if you are interested.

Best,

Dmitri

I am interested, thanks

Hi Lee,

Below is the protocol, which is likely to be very similar to the Cytoskeleton one. Latrunculin and phalloidin/jusplakinolide controls yielded results very similar to those reported by Cytoskeleton.com.

Strictly speaking, this assay does not prevent actin dynamics during cell lysis and spinning procedures. To do that, I would speculate that simultaneous addition of DNaseI and phalloidin to the extraction buffer would be desirable. Phalloidin would prevent depolymerization wherease DNaseI would sequester G-actin and prevent it from polymerization. Pure DNaseI is commercially available from BioWorld.

Best,

Dmitri

11/20/12

F/G actin ratio

1. HeLa Cells – 12-well plate

2. Wash with PBS. Scrape on ice with a plunger from a 1 ml syringe in F-actin stabilization buffer without NaCl, detergents, phalloidin, LatB (12 wells combined in 500ul total final volume).

50 mM PIPES, pH 6.9

5 mM MgCl2

5 mM EGTA

5% (v/v) glycerol

0.1% β-ME

1mM PMSF

10mM benzamidine

3. Pass through 27.5G needle. Incubate for 15 min on ice -> Spin @ 1000g for 5 min @ 4oC.

4. To aliquots of 50ul. Add:

1) Jasplakinolide 1mM-> 10uM final-> 0.5ul/50ul

2) Latrunculin A 2.5mM-> 10uM final-> 0.2ul/50ul

Incubate for 2h on ice.

5. Spin @100,000g for 30’ @4oC.

To 100K supernatant (G-actin fraction), add ¼V (16.7ul) 4xRSB.

To 100K pellet (F-actin fraction), add initial V=50ul of buffer + ¼V=16.7ul of 4xRSB, incubate @4oC for 1h, resuspend by pipetting. Boil, chill on ice.

6. Load 5ul of G and F fractions on 9%PAGE.

Thanks allot!

Lee

Thanks Dimitri, I combined your protocol with the kit materials I have and it worked.

Dear Lee,

Glad to hear that.

Best regards,

Dmitri

Dear Dmitri,

I am also trying differente protocols/buffers to isolate F-actin from HeLa cells. I failed to obtained F-actin in pellet after ultracentrifugation using your protocol. I did some modifications but i think they would not reduce/inhibit F-actin in my samples: Cockt. prot. inhib. (ThermoSci #78429) instead of PMSF and Benz; 6x10^6 HeLas in a 10 cm plate; 10 min on ice after needle passage; no LatB or Jasp controls; 300,000 g, 1 h, 4°C ultracentrifugation.

What do you think may be wrong in this protocol?

Weeks earlier i also failed using commercial kit (Cytoskeleton, Inc. BK037) and another protocols from literature (Saha, 2010 PMID:20181827). Particularly in Saha, 2010 they also use 0.1% of NP40, 0.1% of Triton X-100, 0.1% of Tween 20, 50 mM of NaCl and 1 mM of ATP additionally to the recipe of your buffer described above. In one opportunity i also added phalloidin to their buffer and in a another try i performed whole procedure at 37°C (as in Cytoskeleton, Inc. kit). I failed with both.

Why do you think i am not succeding with any of these protocols?

Thanks in advance.

Best regards,

Alexis

Hi Alexis,

I am not Dimitri, but I used the cytoskeleton kit on HeLa, MCF10A, HCC70 and MDAMB-231 (less recommended) cells. We did not use the HeLa results although I cannot remember any specific problems. I used 6 well plates of high confluence, using the minimal volumes suggested in the datasheet (I think it was 100ul for the F-actin pellet). If you are trying to calibrate the protocol, I would try a cell line that has nice visible stress fibers first, to be sure whether your problem is coming from the protocol or from the cell line.

I ended up using the kit, but did the ultracentrifugation @ 4C and a little longer than the kit required (a colleague from a neighbor lab did 15C).

In the kit you have phalloidin, which i used as a positive control: it pushes the actin equilibrium to increase the amounts of F actin. Then you can see if you are bellow detection amount just because the basal levels of F actin are low, or because your experiment failed technically.

You also mentioned that you didn't perform controls with LatB, but did you use LatB in the F actin sample to disassemble the actin filaments? otherwise they will not run at the right molecular weight because they are still polymerized.

Good luck,

Lee

Dear Lee,

Thanks a lot for sharing your experience.

The Cytoskeleton kit we used was expired at that time but it did not work even after adding a fresh made solution of phalloidin. Do you think there would be another perishable component in this kit totally impaired? (Note: in our experience this kit is not reliable enough mainly because we could not identify their G-actin control (recombinant rat actin) in our blots using their mAb anti-actin or even an anti-actin of ours despite both antibodies can effeciently recognize actin in normal HeLa cell lysates).

I had about 80% confluency and HeLa cells have a nice amount of stress fibers as observed by fluorescence microscopy. I used a volume of lysis buffer (700 uL) that i can say that would give me about 2 mg/mL of protein concentration (measured by Bradford assay).I applied in 20 uL in SDS gels and ponceau staining of supernant was pretty visible and in F-actin fractions was completely absent even in expected mw of actin (44 kDa). Bands intensity in WB of G-actin (supernatant fractions) is high but complely no bands appeared in F-actin (pellet) fractions so i think that i have absolutely no polymerized actin in there. I did not used LatB to depolymerize F-actin fraction but used sample buffer (which has 2-mercaptoethanol 10% and SDS 1%) which i think would be enough to completely disrupt filaments; Dmitri's protocol also uses sample buffer alone. Do you think that absence of LatB in depol. buffer would be the issue?

Thanks again.

Best,

Alexis

Hi Alexis,

Your concentrations etc sound right, I would do the experiment in 6 wells just to save the materials for more experiments. I always had less protein in the F actin samples, I always loaded double vol of that fraction and it was still lower than the G actin.

The company does not mention what are the disassembling factors that they use, but they do supply a depolymerization buffer and the protocol says to incubate it on ice for 1h before adding the sample buffer. I am assuming that it contains some actin disassembling substances like latB and maybe more goodies. I would try to use the kit's dissasembly buffer even with your home made protocol, or use the buffere that was offered in Dimitri's protocol + Jasplakinolide and Latrunculin, incubate for 1h and only then add the sample buffer.

I think that you should use his control as well, it is important because it is a kind of a "whole actin lysate" control.

I noticed now that in the kit they refer to the F-actin enhancing solution as that and not as phalloidin, but once again we assumed it was phalloidin.

I am surprised that you had problems detecting their control actin samples, I actually had the opposite problem, they were so concentrated that they were not comparable to my samples.

Could it be that at some point the kit was mishandled? either during the resuspension and aliquoting process, or maybe even before it reached your lab? Did you already dissolve all the samples? do you keep each reagent in the proper temperature? (4C, -20C, -80C)

Can you ask the local supplier if they had any other complaints about this batch or other products from this particular shipping? (We had such a drama in my institute once).

Anyway, you know that you do get the G fraction and that your AB works. use the F actin enhancing solution as a control, and you should observe a decrease in the amounts of G actin even if you do not detect anything in the F actin.

Hope it will work,

Lee

Hi Lee

I will use LatB in Dmitri's buffer and incubate 1 h in ice before adding sample buffer as you said. Although Cytoskeleton's Depol. buffer is a bit old i will also add LatB and try this one same way. I will also include Jasp. and Lat.B controls as Dmitri does this time. Earlier I said i used fresh phalloidin in Cytoskeleton's buffer but in fact i used fresh ATP at that time. However for next try i can also add fresh phalloidin to see if it helps. By the way, why some protocols use phalloidin and ATP which i would expect to be crucial to stabilize F-actin and others do not but seem equally efficiet?

I was equally surprised when i saw no bands in actin controls even after i loaded 500 ng and observed a band in Ponceau(!!) with expected actin mw. I think it was not mishandled in our lab neither before reaching us although i can not guarantee it 100%. We also kept it in proper temperatures although it was expired at the time we used it. I will try to find and use another recombinant actin in our lab and see if this works in WB although it is a minor point considering my aim with the protocol.

Good idea to track G-actin samples as a control of F-actin amount but my main objective is to isolate F-actin fraction rather than see a exchange in F-/G-actin ratios.

Do you think that DMSO from cockt. prot. inh. (1% final conc.) could interfere somehow with F-actin stabilization buffer and depolymerize F-actin in my samples?

Thanks a lot.

Alexis

The phalloidin is a positive control, not the experiment itself. Basically, in the right environment, actin will spontaneously polymerize anyway.

Im not sure if 1% DMSO is too high, I would assume that it isn't. But the point is to let the actin fibers go through proteolysis, so I am not sure if you should add it before the process is finished. I am afraid that I can't remember what I did (my final experiment with actin was a year ago)- I think that the kit's manual was considering it. I am almost certain that I added protease inhibitors only after the 1h incubation on ice.

Dear Lee,

I ll be trying these tips of you and few other advices i got with Dr. Anna Kashina and let you know if they worked well.

Thanks a lot for you advices.

Best,

Alexis

Hi Dimitri,

I am also facing evaporation of the sample after spin at 1000000g at 37 degrees. So, as suggested by you, I want to try the spin at 4 degree. You have advised to use DnaseI for G actin and phalloidin for F actin stabilization. I would like to know the concentrations of DnaseI and phalloidin one should use for this assay.

Regards,

Sinjini