Table 2.
Buffer X for squid axoplasm experiments (Composition and Instructions)
| Chemical | MW |
M (in stock) |
grams per 25 ml stock |
mM in X |
ml stock in 25 ml Buffer X |
|---|---|---|---|---|---|
| K-aspartate | 171.2 | 1.0 | 4.28 | 350 | 8.75 |
| Taurine | 125.1 | add by wt. | add by wt. | 130 | 0.407 g |
| Betaine | 135.2 | 1.0 | 3.38 | 70 | 1.75 |
| Glycine | 75.07 | 1.0 | 1.88 | 50 | 1.25 |
| MgCl2·6H2O | 203.31 | 1.0 | 5.08 | 12.9 | 0.323 |
| K2·EGTA (adjust stock to pH 7.2 with KOH before bringing to final volume) |
380.4 | 0.1 | 0.9510 | 10 | 2.5 |
| HEPES (adjust stock to pH 7.2 with KOH before bringing to final volume) |
238.3 | 1.0 | 5.96 | 20 | 0.5 |
| CaCl2·2H2O | 147.02 | 1.0 | 3.68 | 3 | 0.075 |
| Glucose | 180.2 | 0.1 | 0.4505 | 1 | 0.25 |
| K2·ATP (adjust stock to pH 7.2 with KOH before bringing to final volume) |
583.4 | 0.2 | 2.92 (0.1167 g/ml) |
1.0 | 0.25 |
Combine aliquots of stock reagents as indicated in the table and check pH. Adjust pH to 7.2 with KOH if needed. Bring to final volume of 25 ml with 20 mM HEPES pH 7.2 and filter on 45µm Millipore filter or equivalent. Store in suitable aliquots at –20 °C until ready for use. We routinely prepare aliquots without ATP, bringing the stock to 20 ml rather than the 25 ml final volume and storing as 0.4 ml aliquots. Immediately prior to use, an aliquot is thawed and ATP added from a concentrate stock. Experimental agents can be added at this point and the aliquot brought to a final volume of 0.5 ml.