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Actin:Actin-Binding Protein Co-sedimentation Assay

 

This method is useful to determine if a protein binds to either G- or to F-actin.  Actin reversibly polymerizes from G-actin to F-actin.  This transformation can be accomplished in vitro by adding salts to G-actin.  G-Actin requires ATP to keep it stable.

Method

1.      Set up reactions in 2ml centrifuge tubes.  Include controls i.e. protein without actin, and actin without protein.  Mark the inside of the tube, so that the position of the pellet can be predicted. This will help extract the supernatant without disturbing the pellet.

 

Example: G actin 5mM
Protein 5mM
10x  KME*  50ml
and make up to 500ml with buffer G (see below).

*  This is the salt solution added to initiate polymerization of actin as a 10x stock.  Many actin binding proteins are pH and/or Ca sensitive w.r.t. binding actin.  This can be tested by altering the composition of the 10salt solution.  Some variants are shown below.

 

2.      Incubate at least 1 hour at room temp.
3.      Centrifuge at  100K in  TL100.2  for 15mins at 20oC
4.      Remove supernatant and transfer to a clean eppendorf.  Then add 500ml sample buffer.
5.      Add 500ml sample buffer to pellet and allow to stand for 10 mins. Resuspend by pipetting up and down, and transfer to a clean eppendorf.
6.      Wash the centrifuge tube by adding 500ml buffer G (see below) then mix with pellet fraction.
7.      Boil samples for 5mins then load 10ml on a gel. 
10x KME   pH6.5 10xKME   pH8.0    10xKM   pH6.5
0.1M Imidazole 0.1M Tris 0.1M Imidazole
500mM KCl 500mM KCl 500mM KCl
10mM MgCl      10mM MgCl 10mM MgCl
10mM EGTA 10mM EGTA
The following is an example of a co-sediment assay.  G2 is a domain of gelsolin known to bind F-actin weakly.  

Lane 1 are the molecular weight markers.  2&3 are the sups and pellets of actin alone, 4 &5 are the sups and pellets of G2 alone, 6 & 7 are the sups and pellets of actin and G2 together.  Note that there is G2 in the pellet  when run with actin (lane7), but not in the absence of actin (lane5).  Also note that there is less actin in the sup with G2 (lane6) than the sup of actin alone(lane2). This is because G2 stimulates polymerization of actin.

Buffer G :-

 

Stock

per litre

per 100 ml of 100x

 

2 mM Tris pH 8.0

1 M

2 ml

20ml

0.2 mM ATP

0.1 M

2 ml

20ml

0.5 mM DTT

dry

77 mg

0.77g

0.2 mM CaCl2

0.1 M 

2 ml

*

0.002% NaN3

dry

0.2 g

2g

*  Add the 2mls of 0.1M Calcium after the 100x buffer G is made up to 1 litre, as it precipitates the ATP if added to the 100x buffer

 

References:-
 
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