Edman Sequencing Analysis

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John Schulze


Phone: (530)752-7327

Email: jmschulze@ucdavis.edu

Edman Sequencing is still the most robust and fastest approach to sequencing the N-terminus of your peptide or proteins. Samples can be analyzed form both PVDF membranes or from samples in a buffer.

There are specific protocols you need to follow if you want to submit samples on a PVDF membrane. This is mainly to minimize Glycine which can interfere with the sequencers.

 

Frequently Asked Questions

We recommend the small pore (0.1 um) pure PVDF membranes from ABI (ProBlott), Millipore (Immobilon PSQ) or Bio-Rad (Trans-Blot). These membranes have a higher binding capacity than large pore (0.45 um) membranes, reducing the risk of protein loss by passage through the membrane and increasing the efficiency of transfer which can approach 100%.

Once a protein of interest has been separated from contaminating species by SDS-PAGE of 2D-GE, the pure protein can be transferred to a PVDF membrane for direct N-terminal sequence analysis. This “micro” purification protocol of SDS-PAGE followed by electroblotting onto a membrane has become the method of choice for structural analysis of proteins of low abundance or proteins difficult to purify by conventional column chromatography or HPLC. Electroblotting onto a PVDF membrane is fast, simple and very efficient. The resulting “blots” are stable at -20C for several months.

The MSF has a detailed protocol for SDS-PAGE/electroblotting derived from published procedures and personal experience. A good general reference is Chapter 3 of P. Matsudaira’s book, “A Practical Guide to Protein and Peptide Purification for Microsequencing” (1993). Factors affecting the efficiency of transfer and quality of subsequent sequence analysis and limitations of PVDF “blots” follow:


Choice of Membrane


We recommend the small pore (0.1 um) pure PVDF membranes from ABI (ProBlott), Millipore (Immobilon PSQ) or Bio-Rad (Trans-Blot). These membranes have a higher binding capacity than large pore (0.45 um) membranes, reducing the risk of protein loss by passage through the membrane and increasing the efficiency of transfer which can approach 100%.


Choice of Transfer Buffer


Blotting at high pH in CAPS buffer is recommended to avoid contamination with Tris and Gly. While some people still prefer the conventional Tris-glycine buffer system, the blotting membrane must be extensively washed with ultra pure water after electroblotting to remove these contaminants. Tris interferes with the Edman chemistry and glycine interferes with the interpretation of sequence data.

Visualization of PVDF-bound Proteins


There are many ways to visualize the transferred protein. Coomassie blue R-250 staining is the most popular (and sensitive) method followed by Ponceau S and Amido Black. Although metal (silver or gold) staining in the gel is very sensitive (1pmole), it should be avoided as no useful sequence information can be obtained from the transferred proteins.

Optimization of Transfer Conditions


Electroblotting is the most critical step in obtaining adequate amounts of “sequencable” protein on the membrane. Careful optimization of the amperage and time required for transfer is important for the maximum recovery of your particular protein. Typically, 20kDa proteins will completely transfer from a mini-gel at 0.5 A in 10 min. Larger proteins (>100kDa) may require 45-60 min. Electroblotting at lower currents generally results in more efficient transfers, but requires longer times. In most cases, proteins of similar size behave in a similar manner during transfer. But, occasionally, a protein’s transfer characteristics will be unusually dependent upon pH, methanol concentration or transfer time.

Limitation of PVDF membranes in Sequence Analysis


It is not uncommon to carefully isolate, purify, and transfer enough protein for sequencing but not obtain any sequence information at all. When this happens, it is very important to ask two questions which MSF can help answer. Was there really enough protein on the blot? Was the protein “artificially” blocked? If the answers to these questions indicate you have isolated a protein which is N-terminally blocked by post-translational modification (over 80% of all eukaryotic proteins are), one must use a different approach (MS analysis) to obtain any sequence information.

Yes


1. To reduce the possibility of amino-terminal blockage of the protein:

Use the highest purity gel reagents possible

  • make fresh stock solutions for important samples
  • age gel prior to running protein
  • reducing agents should be present during electrophoresis

2. Wear gloves. Handle gels and blots only at the edges to minimize background contamination.

3. The concentration of acrylamide in the lower gel (see B. below) can be from 7-18% but 12.5% is the most useful for 10-70Kd proteins.

4. Use a mini-gel system to maximize protein-to-gel ratio and load as concentrated a protein solution as possible without losing resolution. One wants to load the sequencer with as much protein and as little membrane as possible for best results.

5. Run a set of MW markers on each gel to serve as controls in case the unknown protein is blocked. One can sequence the first few residues of a marker band (ie. lysozyme at 14.3 kD) to make sure the gel reagents didn’t chemically block the protein.

6. If identification of “cys” residues is desired, consider red