Protein Edman sequencing experiments on 2D gels
Immunoblotting using polyvinylidene difluoride (PVDF ) membranes is the most commonly used method for Edman sequencing. Protein samples are transferred from a 2D-polyacrylamide (2D-PAGE) gel to a PVDF membrane by electro-transfer, and this membrane loaded with protein can be used directly for protein sequencing. The source of this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillmant, M. Zivi, C. Damerweil, V. Mitschine, eds.
Operation method
Protein Edman sequencing on 2D gels
Materials and Instruments
SDS Sampling Buffer Separation Gel Buffer Concentration Gel Buffer SDS-PAGE Electrophoresis Buffer Move 3.1 Cleveland peptide mapping[5] For more product details, please visit Aladdin Scientific website.
( 1 ) Samples were separated by 2D-PAGE and then stained with Coomassie Brilliant Blue (CBB ) to remove gel fragments containing protein spots.
( 2 ) The proteins in the gel were eluted with an electrophoresis concentrator at a constant power of 2 W for 2 h. After elution, the gel was dialyzed with MQ water for two days and then lyophilized.
a. Cut the blue protein spots from the 2D gel and soak them in MQ water.
b. Add 750 μl of electroelution buffer to a 2 ml centrifuge tube containing protein spots (5-20 gel fragments) and shake for 30 minutes.
c. Place a 12-15 cm long dialysis membrane (small, no. 24, Wako, Osaka, Japan) in 250 ml of MQ water (in a 300 ml beaker) and boil for 5 min. simultaneously wet a small piece of cellophane with MQ water.
d. Close the small opening of the container with the dialysis membrane, open the large opening of the container, fill with the membrane, twist, and close with a clamp (Figure 18-1A). 
e. Attach this container to the electrophoretic concentrator (Fig. 18-1B; Nippon Eido, Tokyo, Japan ). Place the gel pieces containing the proteins on cellophane (the cellophane is attached to the small opening of the container) and add 750 μl of electroelution buffer. Electroelution buffer was added to the ends of the container, which were connected through the buffer so that the protein could be transferred from the small-mouth cellophane to the large-mouth dialysis membrane. Add the electroelution buffer, and the small portion containing the protein should be connected to the positive terminal.
The membrane should be connected to the positive electrode. f. 2W constant power energized run for 2 h.
g. Remove the dialysis membrane, close it with clamps, and dialyze at 4°C with 3 water changes (deionized water) on the first day and 2 water changes (MQ water) on the second day.
h. Place the protein solution in two to six 2-ml centrifuge tubes and freeze-dry overnight.
i. Dissolve the protein in 30 μl of SDS Sampling Buffer. ii.
( 3 ) Proteins were dissolved in 20 W of SDS Sampling Buffer (pH 6.8) and then up-sampled for SDS-PAGE gel electrophoresis. A gel containing 10 μl of Staphylococcus aureus V8 protease (Pierce, Rockford, 1 L ). ( 0.1 μg/μl) and 10 μl of SDS sampling buffer (pH 6.8 ) were overlaid on the protein samples. Electrophoresis was started until the power was turned off when the protein samples and protease converged on the concentrate gel.30 min Digested the proteins and then continued electrophoresis.
a. Secure the glass plates (100 ml X 140 mm X 1 mm) with clamps and 1 mm spacing between the glass plates.
b. Prepare the separation gel solution in a 100 ml beaker. Mix the solution and fill so that the gel surface is 3 cm below the top of the plate (Hint: fill immediately after adding 10% APS and TEMED).
c. Cover the separator with 1 ml of MQ water.
d. Place the gel at room temperature for 40-60 min for polymerization.
e. Pour off excess water in preparation for filling the concentrated gum solution.
f. Prepare a concentrated gel solution in a 100 ml beaker, fill with separator gel, and insert a comb.
g. Allow the gel to polymerize for 20 min at room temperature.
h. Remove the comb, remove the clamp, and remove the silicone strip.
i. Clean the spiking well with a syringe. ii.
j. Mount the glass plate onto the electrophoresis tank and pour in the SDS-PAGE electrophoresis buffer.
k. Proteins are dissolved in 20 μl of SDS Sampling Buffer (pH 6.8), and then the sample is uploaded for SDS-PAGE gel electrophoresis. A solution containing 10 μl of Staphylococcus aureus V8 protease (0.1 μg/μl) and 10 μl of SDS Sampling Buffer ( pH 6.8) was overlaid on the protein samples, and 30 μl of bromophenol blue solution was added.
l. Start electrophoresis until the protein sample and protease converge to the concentrated gel, turn off the power and digest the protein for 30 min.
m. Run electrophoresis at a constant current of 35 mA until the Bromophenol Blue indicator line reaches 5 mm from the bottom.
n. Stop electrophoresis and remove the glass plate.
o. Separate the glass plates with a spatula.
p. Remove the concentrated gel and remove the separating gel.
3.2 N-terminal and internal amino acid sequence analysis, comparative homology analysis of amino acid sequences
( 1 ) Proteins separated by 2D-PAGE or Cleveland method were electrotransferred onto PVDF membranes (Pall, Port Washington, USA) by semi-dry transfer blotting, and then visualized by Caulmers Brilliant Blue staining.
a. Cut a piece of PYDF membrane of the same size as the gel.
b. Cut a piece of 3 MM Waterman filter paper the same size as the gel.
c. Wash the PVDF membrane with methanol for a few seconds, then place the membrane in 100 ml of Blotting Solution C and shake gently for 5 min.
d. Moisten two pieces of blotting filter paper (3 MM) in Blotting Solutions A, B, and C, respectively.
e. Place the separator gel into 100 ml of Blotting Solution C and shake gently for 5 min.
f. Wet the semi-dry transfer blotter with MQ water, place the filter paper moistened in Blotting Solution A on the blotting plate, and place the filter paper moistened in Blotting Solution B on top. Eliminate all air bubbles, if any. Place the PVDF membrane, separator gel, and filter paper wetted with Blotting Solution C in that order.
g. Turn on the power and run at 1 mA/cm2 for 90 min.
h. Wash the membrane with 100 ml of MQ water.
i. Stain the membrane with Coomassie blue for 2~3 min. ii.
j. Decolorize with 60% methanol for 3 min, twice.
k. Wash the gel with MQ water and dry at room temperature.
( 2 ) The stained protein spots or bands were cut off from the PVDF membrane and placed in the upper glass area of the reaction chamber of a gas-phase protein sequencer Procise 494 or cLC ( Applied Biosystems, Foster City, CA ), and Edman degradation was carried out by applying the standard procedure provided by Applied Biosystems. The obtained amino acids (PTH ) were separated by on-line high performance liquid chromatography (HPLC) and identified according to retention time.
( 3 ) The amino acid sequences were searched in Swiss-Prot, PIR, Genpept, and PDB databases by using the web-based FastA program, and the obtained amino acid sequences were analyzed by comparing with the known proteins in the databases.
3.3 Deconfinement of blotted proteins ( see Note 1 )
1. Acetylserine and acetylthreonine
Proteins containing acetylserine and acetylthreonine at the N-terminus are transferred to a PVDF membrane by electrotransfer. The PVDF membrane with the proteins on it is cut off and treated with trifluoroacetic acid at 60°C for 30 min, and then the proteins are sequenced (see Note 2).
2. Formylated groups
Formylated proteins are transferred to PVDF membranes by electrotransfer, and the PVDF membranes are cut and treated with 300 μl of 0.6 mol/L HCl at 25°C for 24 h. The membranes are then washed with MQ water and dried for sequencing.
3. Pyroglutamic acid
( 1 ) Proteins containing pyroglutamic acid at the N-terminus were transferred to PVDF membrane by electrotransfer.
( 2 ) PVDF membranes with proteins were excised and treated with 200 μl of 100 mmol/L glacial acetic acid [containing 0.5% polyvinylpyrrolidone iodine PVP-40] for 30 min at 37°C (see Note 3).
( 3 ) Wash the membrane 10 times with 1 ml of MQ water.
( 4 ) Infiltrate the PVDF membrane with 100 μl of 0.1 mol/L phosphate buffer (pH 8.0) (containing 5 mmol/L dithiothreitol and 10 mmol/L EDTA).
( 5 ) Add 5 μg pyroglutamyl peptidase and treat at 30°C for 24h.
( 6 ) Wash the PVDF membrane with MQ water and dry it for sequencing.