F-Actin Visualization Kit

Hersteller Cytoskeleton
Kategorie
Typ Assay
Specific against other
Menge KIT 300 slides
ArtNr BK005
eClass 6.1 32161000
eClass 9.0 32161000
Lieferbar
Delivery Time
1-2 Weeks
Shipping Temp.
AT
Storage on Arrival
4C
Citations

Arita et al., 2013. A key role for ROCK in TNF-&alpha, -mediated diabetic microvascular damage. Invest. Opthalmol. Vis. Sci. doi: 10.1167/iovs.12-10757.

Asahara et al., 2013. Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin. Diabetologia. doi: 10.1007/s00125-013-2849-5.

Ren et al., 2012. Stimulation of &alpha, 2A-adrenoceptors promotes the maturation of dendritic spines in cultured neurons of the medial prefrontal cortex. Mol. Cell. Neurosci. 49, 205-216.

Spangler et al., 2012. ETS-1/RhC signaling regulates the transcription factor c-Jun in melanoma. Int. J. Cancer. 130, 2801-2811.

faqs

 

Question 1: Do I need to use the fixative included with the F-actin visualization Biochem Kit or can I substitute something else?

Answer 1:  For successful phalloidin staining, F-actin must be fixed and maintained in its native conformation.  Paraformaldehyde (or some derivative, usually at 4% concentration) is an excellent fixative for this purpose.  Methanol is not a good choice for phalloidin staining because F-actin&rsquo, s conformation is lost with that fixative. 

 

Question 2: Which filter settings should I use to visualize rhodamine-phalloidin?

Answer 2:  The phalloidin in the F-actin visualization biochem kit (Cat. # BK005) is labeled with rhodamine, so the fluorescence microscope will require an excitation filter of 535 nm and an emission filter of 585 nm.

 

Question 3: Can I make my own anti-fade media?

Answer 3: The mounting media provided in the kit contains antifade so it&rsquo, s not needed for the kit protocol, however if another antifade is desired we suggest the ProLong&trade, and ProLong Gold&trade, anti-fade reagents from Life Technologies Inc.  Another option is PVA-Dabco, available from Fluka Biochemica.  All of these will work well with fluorescent phalloidin.

You can also make your own anti-fade reagent.  Here are two different recipes.

Recipe 1

250 nM glucose oxidase

64 nM catalase

40 mM d-glucose in water

1% BME

 

These reagents can be combined in Tris or PBS buffer

Tip: Store all components, separately, at -20°, C in aliquots of 10 &mu, l at a 100X concentration. Catalase and glucose oxidase are dissolved in a Tris or PBS buffer and snap frozen in liquid nitrogen. When thawed and stored on ice, aliquots will maintain activity for several hours. Once mixed together use the solution within 1 h.  For consistency, do not refreeze thawed aliquots.

Tip: Add the glucose oxidase last and just before actually using the imaging buffer. This will initiate the first part of the reaction, which depletes the oxygen from solution.

Tip: DTT can be substituted for BME. Use a final DTT concentration of 10mM.  Note, however, BME and DTT have both been demonstrated to have negative effects on some dyes.  This will need to be determined empirically.

Recipe 2

PVA-Dabco

For 25 ml of 2.5% PVA-DABCO (Make 25 ml of PVA-DABCO in 50 ml culture tube to allow ample room for mixing).

1. Using a 50 ml culture tube, weigh 6 gm of glycerol into the culture tube on the balance

2. Add 2.4 gm of PVA

3. Mix well by repeatedly inverting the tube until the PVA is coated with glycerol

4. Add 6 ml of distilled water and repeat mixing until relatively uniform

5. Mix overnight using a rotator at room temperature

6. Add 12 ml of 0.2 M Tris-HCl at pH8-8.5

7. Heat to 50°, C in a water bath with mixing (approximately 30 min)

8. Add 0.625 gm of DABCO and mix well

9. Centrifuge at 5000g for 15 min

10. Remove supernatant, aliquot (suggest 1 ml), and store at -20°, C

The solution may be kept up to 6 months at -20°, C and up to one week at 4°, C before

becoming milky. Do not refreeze.

Stock materials

PVA-polyvinyl alcohol Sigma (P8136)

DABCO- 1, 4 diazabicyclo [2.2.2]octane Sigma (D2522)

Tris-HCl Sigma (T3253)

Glycerol Fisher G33-1

 

To make 0.2N Tris HCl:

500 ml H2O

15.76g Tris HCl

add 7 pellets NaOH

adjust pH to 8.0-8.5 .

 

 

  If you have any questions concerning this product, please contact our Technical Service department at info@hoelzel.de

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Menge: KIT 300 slides
Lieferbar: In stock
Listenpreis: 475,20 €
Preis: 475,20 €
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