Edman Transfer Membrane PVDF Steps
In the Edman degradation sequencing experiment, the protein samples separated by SDS-PAGE need to be transferred to a PVDF membrane to be fixed before proceeding to the next step of sequencing using the Edman degradation method.
Transfer is the step of transferring samples from the protein gel to the PVDF membrane, which is a crucial part of the sample preparation process for Edman degradation sequencing. The quality of the transfer directly affects the results of the sequencing data analysis. Therefore, the Edman transfer step cannot be ignored. To prevent the diffusion of already separated protein bands in the absence of an electric field, the transfer should be conducted promptly. The specific procedure is described as follows:
- 1. PVDF membrane treatment: Take out the PVDF membrane, soak it in methanol for a few seconds, and then place it in CAPS transfer buffer to prevent it from drying.
- 2. Gel treatment: Take out the electrophoresis gel and place it in the transfer buffer for 5-10 minutes.
- 3. Setting up the transfer cassette: Soak the filter paper and sponge in the transfer buffer briefly, then assemble the transfer sandwich in the order of sponge, filter paper, PVDF membrane, gel, filter paper, and sponge, and place it in the mini transfer tank.
- 4. Electro-transfer: Perform electro-transfer under transfer conditions (50V, 100-170mA) at room temperature, with a transfer time typically ranging from 5 to 2 hours (depending on the specific molecular weight of the protein).
- 5. Pre-staining treatment of PVDF membrane: Remove the PVDF membrane and rinse it with deionized water, soak it in methanol for a few seconds, and then proceed with staining.
- 6. Membrane staining: Stain with a dye (such as Coomassie Brilliant Blue) for 30-50 seconds (do not exceed 1 minute), decolorize with 50% methanol (frequently change the decolorizing solution), and finally wash thoroughly with deionized water and air dry.
Biotech Pak provides Edman sequencing services using the Shimadzu Edman sequencing system for researchers and scientific customers.Protein N-terminal sequencing based on Edman degradationservices. Using our sequencing system, you can determine the sequence information of the first 30 amino acids at the N-terminal. By using a specific protein loading system, you can determine 60-70 amino acids at the N-terminal. Biotech Pak has also established a platform for N-terminal sequencing using advanced LC-MS/MS technology, capable of identifying blocked and modified protein termini, whichcomplements protein N-terminal sequencing based on Edman degradationto ensure smooth N-terminal sequencing services.
Related services:
Protein N/C terminal sequencing
Biopharmaceutical N/C terminal sequencing
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