Electroblotting

 

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 eucaryotic proteins are), one must use a different approach (MS analysis) to obtain any sequence information.

Please click here if this helped you.
7 people found this helpful.


Category: Edman Sequencing

← Electroblotting
Posted in