Electrobloting of Proteins on PVDF for Sequencing

SDS-PAGE ELECTROPHORESIS

Essentially SDS-PAGE is carried out as usual with the following considerations taken into account for potentially maximizing sequencing success. Most of these considerations are designed to decrease the incidence of artifactual N-terminal blockage during electrophoretic manipulations, and thus increase the available amount of sequencable protein.
1. Try to start with a minimum of 100 pmoles of your protein (this would be about 3 ug of a 30kD protein). There is a wide range of blotting efficiency which is dependant on the individual protein, the blotting buffer and membrane used, blotting time, etc. If only 50% of your protein successfully transfers and binds the membrane, and if the initial yield of N-terminal amino acid is only 50%, then sequencing would be in the range of ~25 pmols which is detectable with this system. If your protein stains well with Coomassie Blue on the membrane after transfer, there is probably enough to sequence (providing it is not blocked).
2. Try to use a minigel to keep the acrylamide:protein ratio as low as possible. The gel should be cast (including the stacker) and aged for 24-72 hours at room temperature to help decrease some of the compounds which may artifactually block the N-terminus during electrophoresis. Use only the highest quality reagents.
3. Other hints that may help prevent artifactual blockage of the N-terminus (according to the BioRad Technical Bulletin on PVDF membrane, based on Speicher, D.W.; in Techniques in Protein Chemistry (T. Hugli, Ed.), Academic Press (1989), p. 24-35.) :
  1. Do not use urea in the sample solubilizing buffer, and heat the samples at 37ºC for 10-15 minutes rather than 100ºC prior to loading on the gel. An example of a 5X Sample Solubilizing Buffer:
    
    0.5M sucrose (or 50% glycerol)
    15% SDS
    312.5 mM disodium EDTA
    
    Heat gently to get into solution and adjust pH to 6.9 with HCl. Store at 4ºC. Add 100 ul of βmercaptoethanol and 100 ul of bromophenol blue solution (0.05% w/v) to 2 ml of the 5X stock prior to use.
  2. Add 11.4 mg/liter (0.1 mM) thioglycolate to the upper running buffer prior to electrophoresis to scavenge reactive compounds left in the gel.
ELECTROPHORETIC TRANSFER

Tank blotting is usually preferable to semi-dry blotting because it tends to be more quantitative with higher binding yields
1. Remove the gel and soak in transfer buffer to remove SDS and glycine. Do not soak for too long or some diffusion may occur and there may be increased difficulty in transferring larger proteins. Beckman (Porton) recommends first soaking the gel in deionized water containing DTT (at 2mg/100 ml), followed by two 5 minute washes in the transfer buffer, also containing 2 mg DTT/100 ml.
2. Transfer Membrane: PVDF (polyvinylidene difloride) membrane is most commonly used for blotting when the protein is to be directly subjected to sequencing, due to its protein binding properties, mechanical strength, and resistance to the chemicals used in the sequencing protocol. PVDF is extremely hydrophobic however, and therefore must be initially wetted in methanol (or another suitable solvent) for several seconds until translucent. Immediately transfer to the blotting/transfer buffer which you will be using in order to equilibrate the membrane (this should only take a few minutes and you will note the membrane becomes submersible in the buffer). Do not allow the membrane to dry before blotting begins, or you will have to re-soak in methanol and re-equilibrate in buffer. Several companies supply PVDF membrane for blotting - among them are Millipore (ImmobilonP and ImmobilonPSQ), Beckman/Porton (Hyperbond), ABI (Problot), and BioRad (Trans-Blot). ImmobilonPSQ is supposed to have higher binding and less "blow-through" compared to ImmobilonP and may be better for smaller proteins.
3. Transfer Buffer: There are several buffers which are used for transfer of protein onto PVDF for direct microsequencing. In general, SDS is avoided because it causes increased loss of the protein from the membrane during sequencing, however its absence may decrease transfer efficiency, particularly of larger proteins. Glycine is also often eliminated from the transfer buffer because of the high background glycine peaks which then occur during sequencing, particularly in the earlier cycles. Following are some buffers which have apparently been used successfully, with CAPS Buffer probably being the most common.
  - CAPS BUFFER
    - Matsudaira, ((1987). Sequence from picomole quantities of proteins electroblotted onto PVDF. J. Biol. Chem. 262:10035.
    - 10X Stock CAPS 100 mM CAPS, pH 11.0
      (3-[cyclohexylamino]-1-propanesulfonic acid)
      dissolve 22.13 g CAPS in 900 ml highly purified water; titrate with NaOH to pH 11.0 and fill to 1 liter; store at 4 C.
    - 1X CAPS BUFFER 10 mM CAPS, 10% Methanol
      prepare 1 liter by mixing 100 ml of 10X Stock CAPS with 100 ml methanol and 800 ml water
    Assemble the blotting "sandwich" and blot at 50 volts (~0.15 A), 30-60 minutes, room T or
    90 volts (0.3 A),15-30 minutes, room T or
    0.5 A, 10-30 minutes, room T
    
    These values are taken from several technical bulletins and are presented to give an approximate set of values from which to begin. Actual blotting conditions will depend on the individual protein of interest, with, in general, longer times (and/or) greater current required for larger proteins.
  - TOWBIN BUFFER
    - Towbin, H., et al. (1979). PNAS USA 76: 4350.
    - 10X Stock TOWBIN 250 mM TRIS, pH 8.3
      1.92 M glycine
    Transfer buffer is 0.5X Towbin with 10% metanol (and no SDS).
    
    Assemble the blotting "sandwich" and blot at 40 volts (0.3 A), 1-4 hours, rm T
    These values are only meant as an example, with the conditions dependent on the individual protein of interest.
  - TRIS-BORATE BUFFER
    - Bauw, G. et al (1989). PNAS USA 86:7701-7705.
    Transfer Buffer 50 mM Tris base, pH 8.5 (with NaOH)
    5 mM borate
    
    The gel was equilibrated 30 minutes in this transfer buffer prior to blotting. Some proteins may transfer faster than with the CAPS buffer system, but some proteins (especially smaller ones) may "breakthrough" the transfer membrane (although this did not appear to be a problem with all membranes). This buffer system also appeared to give a higher sequencing background than CAPS (see Baker, C. et al (1991). Electrophoresis 12: 342-348.)
  Methanol (10-20%) added to the blotting buffers may improve the binding efficiency of low molecular weight compounds, but may also cause poorer transfer of high molecular weight components due to "fixative" effects of methanol. CAPS appears ,in general, to give a more uniform transfer onto the hydrophobic PVDF membrane, however, the high pH of this buffer may cause cyclization and deamidation of labile peptide linkages (s.a. ASN-GLY) (Robinson, A.B., et al.(1970). PNAS USA 66: 753.). Much of this information was taken from Baker, C., Dunn, M., and Yacoub, M. (1991). Evaluation of membranes used for electroblotting of proteins for direct automated microsequencing. Electrophoresis 12: 342-348.
4. Protein Staining: After blotting, the PVDF membrane is rinsed in highly purified water for 5 minutes (Beckman suggests adding 2 mg DTT/100 ml). If blotting was done in Towbin buffer, the membrane may be washed in three 5 minute water washes to help remove the extra contaminating glycine. There are several stains which are compatible with direct microsequencing, with the most common being Coomassie Blue R-250. In general, staining and de-staining times should be kept to a minimum.
  1. COOMASSIE BLUE R-250
    
    - 0.1% Coomassie Blue R-250
    - 40% methanol
    - 1% acetic acid
    
    Stain, with shaking, about 1 minute.
    
    ABI (ProBlot) recommends soaking the membrane in 100% methanol for a couple of seconds after the water wash and prior to the staining step.
    BioRad suggests decreasing the Coomassie to 0.025% and increasing the staining time to about 5 minutes. They also recommend eliminating acetic acid from the stain due to potential blocking of the N-terminus (although I have seen some recipes with up to 10% acetic acid and with no apparent problems) The membrane is immediately de-stained with 50% methanol, either several changes of 1-2 minutes each, or for 10-15 minutes, or until the background is light blue (the membrane gets lighter as it dries). The membrane may now be washed in water (5-10 minutes), air dried, and stored at room temperature, or the band of interest excised with a clean, sharp razor blade.
  2. AMIDO BLACK (from ABI (ProBlot) Technical Bulletin)
    
    - 0.1% amido black
    - 40% methanol
    - 1% acetic acid
    
    Dissolve 1.0g amido black in 400 ml methanol. Stir at least 1 hour. Add 10 ml acetic acid and 590 ml water. Stir an additional 30 minutes. Filter through 45 u filter.
    
    Stain as with Coomassie above, except de-stain in water.
  3. PONCEAU S (from ABI (ProBlot) Technical Bulletin)
    
    - 0.2 % Ponceau S
    - 1% acetic acid
    
    Dissolve 0.4 g of Ponceau S in 198 ml of water and stir 30 minutes. Add 2 ml of acetic acid to the mixture.
    
    Stain as with Coomassie above (about 1 minute), except de-stain with water (sometimes de-staining is not even required).
COMMON N-TERMINAL BLOCKING GROUPS:

It has been estimated that up to 80% of eukaryotic proteins are blocked at the N-terminus and therefore refractory to direct sequencing. Some common blocking groups are:
- pyroglutamic acid - can be removed with pyroglutamyl amino peptidase.
- N-acetyl groups - commonly found on alanine,serine, methionine, glycine, threonine.
- formyl group - on methionine
If enough pure protein is available, some amino acid sequence can still be obtained, even from blocked protein samples. This can be done by cleaving the protein into smaller peptides either enzymatically (e.g. trypsin) or chemically (e.g. cyanogen bromide). The fragments can be isolated (by HPLC or re-blotting and selected peptides analyzed for their N-terminal sequence. This can provide some amino acid sequence from internal sites within the protein. (Aebersold, R.H., et al.(1987). Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. PNAS USA 84: 6970-6974.)

Center for Biotechnology and Genomics

Texas Tech University

Electrobloting of Proteins on PVDF for Sequencing