Native gels with actin monomers
Jamie Moseley (3/13/05)
Solutions:
5X Buffer (1L)
15.2g Tris base
72.8g glycine
1L ddH2O
Running buffer (1.L)
300ml 5X buffer
0.17g ATP
7.5ml 100mM CaCl2
300µl 1M DTT
1200ml ddH2O
3.3X loading buffer (2ml)
1.2ml 5X buffer
0.6ml 50% glycerol
12µl 0.1M ATP
33µl 100mM CaCl2
1.32µl 1M DTT
7.5% gels (20ml)
4ml 5X buffer (1X final concentration)
40µl 0.1M ATP (0.2mM final)
200µl 10% TX-100 (0.1% final)
5mL 30% acrylamide (7.5% final)
100µl 100mM CaCl2 (0.5mM final)
4µl 1M DTT (0.2mM final)
10.5ml ddH2O
**100µl 10% APS
**20µl Temed
**: add immediately prior to pouring gels.
Method:
1. To pour gels, use 1mM spacers and 1mM combs (large wells, 10 wells/comb). All other
plates and gel parts same as SDS-PAGE gels. These gels have no stack, so fill to top and
add combs. Once poured, these gels should be used within several hours and cannot
be saved as with SDS-PAGE gels.
2. Prepare samples by combining proteins in 10 or 20µl reactions (10µl easier to load). 3.3X
loading buffer should be added to a 1X final concentration. Proteins are generally mixed
at 1:1 ratio, and using higher concentrations will increase probability of seeing visible
shift in migration. Be sure to include appropriate controls, i.e. actin alone (in the absence
of your protein of interest), and your protein of interest alone (in the absence of actin).
The following is an example to test binding of cofilin (50µM stock in 10mM Tris) and Gactin (20µM stock in G-buffer) at a final concentration of 10µM each in 10µL reactions;
this reaction provides a good positive control.
Reaction
Actin alone
Actin+Cofilin
Cofilin alone
3.3X buffer
3µl
3µl
3µl
G-actin
5µl
5µl
0
Cofilin
0
2µl
2µl
G-buffer
0
0
5µl
10mM Tris
2µl
0
0
3. Let reactions sit on bench ~10 min. prior to loading onto gel to allow binding. In general,
2 gels are loaded with the same samples (in duplicate). For this reason, it is easiest to set
up 20µl reactions (10µl for each gel). Gels are loaded similar to SDS-PAGE gels;
however, there is no blue dye in leading buffer, so use caution when loading as it is
difficult to visualize sample.
4. Gels are run at constant voltage (100 Volts), NOT constant amps. Run one gel for
approximately 80 min. Continue running the second gel for an additional 70 min. (150
min. total). Coomassie staining the first gel will give you an idea how long to run the
second gel. You will observe a better migration shift if samples run longer; however, you
want to keep your proteins from running off the gel. Remember, there is no dye front to
signal when gel is “done.” Additionally, samples run according to charge—not size—so
you likely don’t know where your protein will run. Therefore, use your coomassiestained first gel as a measure for how long to run the second gel.
5. Gels may be stained with Coomassie or transferred for western blotting similar to a SDSPAGE gel.