1. It is recommended to thaw the protein samples only once, and discard the rest. Repeating thawing-freezing rounds may generate artifacts due to protein degradation.
2. We also use gradient gel 5-15% polyacrylamide that gives a good protein separation. Casting of such a gel may represent some difficulties. Precasted gradient and standard gels are also commercially available.
3. Caution: when pouring acrylamide solution, plan to have the separating gel at least 0.5 cm below the slots.
4. 10 teeth-comb (1 mm) allows the loading of maximum 25 |L or up to 200 |g of protein extract per slot, without aberrant migration of the proteins, while 15 teeth-comb (1 mm) allows loading of maximum 15 |L or up to 50 |g of protein extract per slot.
5. For the gel prepared with the MINI-PROTEAN II apparatus, nitrocellulose membranes of 9 cm x 6.5 cm will be large enough for the protein transfer. They should be carefully handled with gloves.
6. SDS is usually dispensable for the transfer, but we consistently observe a better immunodetection with 1C2 antibody when the transfer was performed with SDS.
7. The antibody concentration to be used on WB may vary from one batch to another and should be determined, but 1/2000 is a good dilution to start with.
8. Using incubation for 1 h at RT, we obtained better detection and less background than when the detection is performed overnight at 4°C. This is thought to be due to the instability of the 1C2 antibody once diluted.
9. The antibody solution should cover the membranes during rocking to keep them wet. Alternatively, the membranes and antibody solution can be sealed in a plastic bag.
10. In general, antibody solution can be reused immediately for incubation with other membranes or after being stored overnight at 4°C. Our experience indicates that repeatedly freezing and thawing the diluted 1C2 antibody decreases the polygln expansion detection and increases the background.
11. The secondary antibody should be diluted according to the supplier's instructions; too high a concentration of the secondary antibody would result in high background.
12. Given the very stable interaction of 1C2 with long polygln, intensive washes are strongly recommended to reduce the background as much as possible. Washing can even be extended to several hours without loss of signal.
13. If background is high the membrane may be rewashed and redetected, but this causes a slight loss of sensitivity.
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