|CNBR Digest of Protein||Electroblotting of Proteins on PVDF for Sequencing|
|Oligo Purification Using PolyAcrylamide Gel||In Gel Digestion of Proteins Separated by PAGE|
(based on method in "A Practical Guide to Protein and Peptide Purification for Microsequencing", 2nd Ed.,Paul Matsudaira ,Editor, Academic Press, N.Y., 1993)
- Suspend protein in 70% formic acid. (note: Formic acid is usually obtained as an 88% solution). We have used up to about 100mg protein per 100 ml of formic acid.
- Make up a 70 mg/ml or 0.66M CnBr stock solution: 132 ml of 5M CnBr in acetonitrile and 868 ml of 70% formic acid. CnBr is very dangerous and should be handled accordingly.
- Add 10 ml of the 70 mg/ml CnBr solution per 10 mg of protein.
- Incubate in the dark at room temperature for 24 hours.
- Speed-Vac the the tube contents to dryness. Add an additional 100 ml of water to the tube and re-dry in the Speed-Vac.
- The protein should now be cleaved at Met residues, and the resulting peptides may be separated on HPLC or by PAGE depending on the size and amounts of the peptides.
Electroblotting 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.
- 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
- 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.
- 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.) :
- 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)
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.
- 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.
- 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:
- 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).
- ELECTROPHORETIC TRANSFER
Tank blotting is usually preferable to semi-dry blotting because it tends to be more quantitative with higher binding yields
- 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.
- 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.
- 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.
- 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
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, 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.)
- CAPS BUFFER
- 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.
- 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.
- 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.
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).
- COOMASSIE BLUE R-250
- 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.
- 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
The quality of an oligonucleotide (ie. how much is full-length product versus
incomplete products) may be assessed by loading approximately 5 ug on a 19 or
20% polyacrylamide/urea minigel and staining with ethidium bromide. This same
gel system may also be used in a preparative manner to purify the full-length
from the incomplete oligonucleotide products.
Recipe for 19% acrylamide/urea gel
This recipe is for 30ml, which is enough for a standard size gel. You need only 5-10ml for a typical minigel (for quality assessment or for purifying only a portion of the oligonucleotide). There is no stacking gel in this gel system.
- 15 ml of 40% acrylamide (1:22)*
- 14.4 g urea >
- 3 ml 10X TBE
- warm to 37o to dissolve urea
- Add water to fill to 30ml. Just before pouring gel, add:
- 120 ul of 15% ammonium persulfate
- 20 ul TEMED
- Pour the gel. Put in the appropriate comb (either a "welled" comb
or preparative style), and allow the gel to polymerize at a 20º angle. Add more acrylamide
solution to the top of the gel around the comb during polymerization if
- Pre-run the gel in 1X TBE for 1 hour at 200V (minigel) to 400 V (regular size gel).
*(1:22) 40% acrylamide
Fill to 60ml with water. This may be stored dark, 4ºC for several weeks after filtering and degassing.
- Running the Sample
- Prepare the sample by dissolving the desired amount in water and adding an equal volume of formamide. Load one lane with xylene cyanol/bromophenol blue, with an equal volume of formamide, as marker dyes (these run approximately equivalent to an 8 mer and 28 mer oligo in this gel system). Heat samples and markers to 65o for 10 minutes. Clean out the wells by gently squirting with a pasteur pipet until unpolymerized material is gone, and load the samples. If running a quality assessment gel to be stained with ethidium bromide, use approximately 5-10ug of material, with the final volume of sample (with the formamide) being appropriate for the well size of the gel you are using. If running a preparative gel for purification, load about one third to one half of the sample into the preparative well, keeping the final volume with the formamide appropriate for the well volume. Use a separate well to load the marker dyes.
- Run the gel under the same conditions as pre-running.
- When dyes have migrated the desired distance, remove the gel and either stain in ethidium bromide or wrap in saran wrap for ultraviolet shadowing (for the purification gel). Lay the wrapped gel on a treated silica plate (fluoresces at 254nm) and illuminate with a handheld UV illuminator at 254nm (short wave). The DNA will absorb the light and appear dark against the fluorescing silica. Work quickly, and using a clean razor blade cut out the largest (full-length) dark band. Cut the acrylamide strip into small bits and transfer to a tube(s).
- Add 1 ml of 0.3M Na0Ac per 2 cm length of gel slice. Agitate, if possible (improves elution of oligo), at 37ºC overnight. Spin down acrylamide and carefully transfer supernatant and respin to ensure all acrylamide bits are removed. Extract the supernatant with an equal volume of phenol:chloroform (1:1). Precipitate the oligonucleotide with 2 volumes of ethanol at -20ºC. (If sample will not precipitate, use 6 volumes of acetone or a mix of ethanol and acetone.) Wash the pellet with1 ml of 95% ethanol, dry, resuspend, and determine the concentration of oligonucleotide spectrophotometrically. You should be able to recover approximately 100-400ug of oligonucleotide per 200nmol synthesis with this method.
In Gel Digestion of Proteins Separated by PAGE
In Gel Digestion of Proteins Separated by PAGE (According to EMBL Protein and
Peptide Group, 1997)
Extracted from: http://www.pharma.ethz.ch/bmm/protocols/ingeldig.html, with some modifications.
- Excision of protein band or spots from polyacrylamide gels:
- Rinse your gloves with water to avoid traces of dust in the sample.
- Rinse the gel with water.
- Excise spots with a clean pipette tip cut off (to the correct diameter) or with a razor blade, cutting as close to the edge of the spot as possible (get as little background gel as possible). Cut into small particles.
- Transfer gel into an Eppendorf tube.
- Reduction and Alkylation:
- Wash the cut-up particles with 100-150 ul of water (5 min), spin down (table-top centrifuge, max. 2800 rpm) and remove liquid.
- Add acetonitrile (3-4 gel volumes) and wait 10-15 min, spin particles down and remove all liquid, dry down in a speed vac.
- Swell the gel pieces in 10 mM DTT/0.1 M NH4HCO3, enough to cover the gel, and incubate for 30 min at 56ºC (water bath) to reduce the protein.
- Spin down and remove excess liquid, shrink with acetonitrile.
- Replace acetonitrile with 55 mM Iodoacetamide/0.1 M NH4HCO3, incubate 20 min at room T in the dark.
- Remove iodoacetamide solution, wash the gel particles with 150-200 ul 0.1M NH4HCO3 for 15 min.
- Spin down, remove all liquid and shrink with acetonitrile.
- Remove all liquid and dry gel particles down in speed vac.
- Washing Gel Pieces to remove unpolymerized acrylamide and contaminants:
- Wash the sample 5 min in 80 ul 0.1 M NH4HCO3
- Add an equal volume of acetonitrile and vortex 5 min.
- Spin down the particles and remove the solution.
- Repeat this wash step twice.
- Shrink the gel pieces with acetonitrile about 5 min
- Remove the solution and dry down in a speed vac.
In-gel digestion with trypsin:
- Pre-cool tubes with the dried gel particles and the trypsin on ice.
- Rehydrate the gel particles in the trypsin solution (20 ng/ul Trypsin(Promega #V5111) in 25 mM NH4HCO3) for 30-45-min at 4ºC. Add just enough solution to cover (~20 ul) the gel pieces.
- After 15 min, check the sample and add more trypsin solution if the solution has been completely absorbed by the gel pieces.
- After a total of 30-45 min rehydration, remove the remaining trypsin solution.
- Add 10-75 ul (to just cover) 25 mM NH4HCO3 without trypsin and incubate 16-20 hours at 37ºC.
- Spin down the condensed water droplets.
- Transfer liquid supernatant which can already contain small peptides (<2000 Da) to a fresh 0.5 tube. Keep on ice.
Extraction of Peptides from Gel:
- Add 10-15 ul of 25 mM NH4HCO3.
- Incubate 15 min at 37ºC while shaking (vortex every 5 during incubation)
- Spin down and add acetonitrile (1-2 times the volume of the gel ~10-80 ul).
- Incubate 15 min at 37ºC while shaking.
- Sonicate for 5 min at 37ºC.
- Spin down liquid and transfer the peptide containing solution to the same tube saved before containing peptides.
- Add 10-50 ul of 5% formic acid (to adjust pH).
- Incubate 15 min at 37ºC while shaking.
- Spin down and add acetonitrile (1-2 times the volume of the gel).
- Incubate 15 min at 37ºC while shaking.
- Sonicate for 5 min at 37ºC.
- Spin down and transfer the peptide containing solution to the same tube as before, with basic extracted peptides.
- Dry in a speed vac.