Rapid Turnaround Protein Identification by MALDI-TOF/TOF
Please follow these tips when cutting bands/spots from your gel
- Always wear fresh gloves when handling gels and change them frequently.
- Precast gels are strongly recommended along with MS compatible stain kits. Stain for the minimum time.
- Wash the gel thoroughly three times with deionized water in freshly rinsed new petri or square dishes.
- Cut the gel band/spot in a clean hood. Use a different pre-washed scapel blade for each band or wash with water and methanol before using it to cut the next band.
- Transfer each cut band/spot to a “cleaned” labeled eppendorf tube, cap and bring to the Proteomics Shared Facility.
Important Information When Preparing your SDS-PAGE Gel Bands or 2D gel spots
Automation for rapid protein identification of the most abundant proteins identified in a gel band/spot has been set up in the lab since April, 2009. To minimize client-introduced contaminating proteins, it is important to follow these procedures. In most cases, identifying the most abundant protein will be sufficient. If needed, further types of proteomics analysis is possible, e.g., nanoLC-MS/MS. Controls to check for contamination during sample handling on our end and for sensitivity will be included. If no protein is identified from the gel spot/band there will be no charge. If client-introduced contaminating proteins are detected, the $50 charge per sample/spot will still apply. An additional cost saving feature is available for those performing their own digestion. In these cases we can provide a MALDI target for the samples to be spotted. Data acquisition and protein identification will be usually performed within 48-72 hours.
Contaminants: The most common contaminant in SDS-PAGE gels is keratin. Hence, it is necessary to ensure that precautions are taken to minimize contamination, such as from hair and skin.
Gels: Precast gels are strongly encouraged, along with freshly prepared solutions when necessary.
Stains: Mass spectrometry compatible stains are required, such as GelCode Blue or Silver Stain II, both available from Pierce. Staining for a short time is also recommended.
Cleanliness: Rinse all glassware, containers, pipets and other equipment, including gloves, with deionized water that you will come in contact with while cutting your gel band/spot.
Tubes: The eppendorf tube that you will place you gel band/spot must be free of any interfering chemicals, e.g. silicone, detergents, salts. These tubes must first be rinsed with deionized water followed by 100% acetonitrile or methanol (HPLC grade) and allowed to air dry in a clean hood. BioRad tubes with rinsing works well (1.5 mL Catalog # 223-9480).
Controls: We need a negative control band or spot from your gel.
Come see us to get started!