Procedure

To ensure that polyacrylamide gel cast using the WIDE RANGE Gel preparation buffer (Nacalai Tesque, Prod No. 07831-94) are applicable to mass spectrometry analysis, the gel was compared with the conventional Laemmli PAGE gel using the procedure described below:

1. Bovine serum albumin (BSA) was loaded in each WIDE RANGE and Laemmli gel.
2. Gel staining was then performed on both gels using the Gel Negative Staining Kit (Nacalai Tesque, Prod No. 16660-41).
3. The protein bands indicating BSA were then excised from each gel, after which trypsin digestion was performed using the In-Gel Tryptic Digestion Kit.
4. Dialysis was then performed on each sample to eliminate residual salts, after which MALDI-MS analysis was conducted using LTQ-XL.
   

<MALDI-MS condition>

Matrix: 2,5-Dihydroxybenzoic Acid (DHB)
Mode: Positive mode
Range: m/z 800-4000
Laser energy: 30µJ
 

Conclusion

We found that the appropriate peptide peaks could be detected in the BSA bands excised from the SDS-PAGE gels cast by both the WIDE RANGE and Laemmli protocol gel. Furthermore, because the WIDE RANGE gel offers significantly more tensile strength than the Laemmli protocol gel, it facilitates the handling of subsequent steps.