I

Protein database searching for identification of carbonylated proteins

Fig. 11.1. Protocol to identify carbonylated proteins using 2D-gel electrophoresis and western blotting.

2. Determine the protein concentrations in the sonicated samples by using BCA reagent kit.

3. Approx 100-150 pg of the protein is used to derivatize the samples with DNPH for detection of protein carbonyls.

4. To derivatize the samples, add DNPH four times the volume of sample, vortex the samples and incubate at room temperature for 20 min without shaking.

5. Add 100% TCA to the samples to get a final concentration of 30% TCA to precipitate the protein. Incubate the samples on ice for 10 min.

6. Centrifuge the samples at 10,000g for 5 min at 4°C.

7. Decant the supernatant and wash the pellet four times with ice-cold ethanol/ethyl acetate (1:1) mixture. (see Note 1).

8. All the washing steps should be carried out at 10,000^ for 5 min at 4°C.

10. Suspend the pellet in 200 pL of IEF rehydration buffer and incubate at room temperature on a vortex for 1-2 h.

11. Sonicate the samples for 10 s before loading the samples in IEF tray (BioRad).

12. Samples for the protein expression are processed as mentioned previously for the DNPH derivatization except that instead of DNPH, 2 N HCl is added.

3.2. IEF of Samples 1. IEF is performed with a BioRad system using 110 mm, pH

or First Dimension 3-10-immobilized pH gradient (IPG) strips.

2. Transfer 180 pL of the samples into the bottom of the well in IEF tray carefully by using a micropipet. Avoid bubbles.

3. Pick up the IPG strips with forceps and remove the plastic sheet from the IPG strips. Place IPG strips on top of the sample with gel side facing down. Make sure that the +ve end of strip is towards the +ve end of the IEF tray. This is important for proper operation.

4. Cover the IEF tray and place the IEF tray in the IEF machine and close the lid of the IEF machine. IPG strips are actively rehydrated at 50 V (20°C) overnight.

5. After 1 h open the lid of the IEF machine without turning off the instrument and take the IEF tray outside the machine and add 2 mL of mineral oil in each well to cover the IPG strips (see Note 3).

6. Place the IEF tray in the IEF machine and close the lid and carryout the active rehydration step for about 16 h.

7. After 16 h, wet the paper wicks with 8 pL of nanopure water and lift the IEF strip using forceps and place a paper wick on both the electrodes (see Note 4).

8. Place the IEF tray into the machine with right electrodes connection and carry out isoelectrofocusing at 20°C as follows: 300 V for 2-h linear gradient, 500 V for 2-h linear gradient, 1,000 V for 2-h linear gradient, 8,000 V for 8-h linear gradient, and 8,000 V for 10-h rapid gradient.

9.After completion of IEF the IPG strips are transferred to equilibration tray and either processed directly for second dimension or stored in -80°C freezer until use (see Note 5).

3.3. Two-Dimensional Electrophoresis (Separation Based in Migration Rate)

1. Heat agarose solution (see Note 6)

2. Take the IPG strip out from the -80°C freezer and thaw the IPG strip at room temperature (IPG strips color change from milky white to clear)

3. Incubate the IPG strips in 4 ml of equilibration buffer containing DTT in a disposable equilibration tray with lid with the gels side facing up for 10 min. Keep the equilibration tray in dark (see Note 7)

4. While waiting for equilibration, prepare 2D-gel by rinsing the gels with DI water (inside and outside). And also prepare 1X running buffer by diluting 100 mL of 10X buffer with 900 mL of deionized water in a measuring cylinder

5. Remove white plastic tape from the bottom of the 2D gels

6. Remove extra water with Kimwipes (tissue paper)

7. After 10 min, transfer the IPG strips into next well in the equilibration tray and add 4 mL of equilibration buffer containing IA, again with the gel side facing up for another 10 min. Keep the equilibration tray in dark (see Note 8)

8. Dip the IPG strips into 1X running buffer to remove excess equilibration buffer

9. Place the IPG strips with gel side facing up into criterion gels. Do not push the IPG strip into the well

10. Load unstained molecular weight marker into standard well adjacent to the IPG strip well to stain the gels and load precision stained molecular weight markers on the gel that will be used for oxyblot

11. Add warm agarose solution into the wells of criterion gels, avoiding bubbles, and then slowly push the IPG strip on either end till a contact is established between the gel and IPG strip (Strip must be in parallel contact with the gel)

12. Wait for 10 min for agarose to solidify and then place the gels in tank filled with running buffer and then fill the upper tank with running buffer

13. Connect power supply with right connection (+ve to +ve and -ve to -ve)

14. Run the gels at 200 V for 65 min at room temperature, until the dye front (bromophenol blue) exits the gel into the lower tank

15. Disconnect the power supply and disassemble the 2D-

apparatus. The gels plates are broken open to remove the gels.

3.4. Protein Staining

3.5. Oxyblot (Immunochemical Detection)

1. The gels containing non-derivatized proteins with unstained marker is fixed in 50 mL of fixative solution at room temperature for 60 min with gentle agitation.

2. Remove fixative solution and add 50 mL of SYPRO Ruby gel stain and incubate overnight at room temperature on a rocking platform.

1. Gels containing DNPH derivatized proteins are transferred to nitrocellulose membranes for immunochemical detection of protein carbonyls

2. The samples that have been separated by SDS-PAGE are transferred to nitrocellulose membranes using a semidry transfer unit. The gels, nitrocellulose membrane and filter papers are soaked in cold transfer buffer for 10 min.

3. A setup of transfer is prepared in the following order: first place one soaked filter paper on the transfer unit platform, followed by nitrocellulose membrane, gel, and one more filter paper. Roll a glass rod after the gel is placed onto the nitrocellulose membrane to remove bubbles that are trapped between the nitrocellulose membrane, gels, and filter paper. Once the sandwich is ready roll the glass rod once again to ensure no bubbles are is trapped in the transfer sandwich.

4. Connect power supply with right connection (+ve to +ve and -ve to -ve). It is critically important to ensure correct orientation of the power supply or the proteins will move from gel into the filter paper instead of the nitrocellulose membrane.

5. Close the lid of the transfer unit and activate power supply. Transfers are performed at 15 V for 2 h at room temperature. The semitransfer method of proteins not only saves time, but it also requires less amount of transfer buffer.

6. Once the transfer is completed, the transfer unit is disconnected and the unit is carefully disassembled, and the nitrocellulose membrane is taken out. Since pre-stained molecular weight markers are used, we do not need to cut the ends of the nitrocellulose membrane for orientation. The gel and filter papers can then be discarded. The pre-stained molecular weight markers should be clearly visible on the membrane.

7. The nitrocellulose is then incubated in 50 mL of blocking buffer for 1 h at room temperature on a rocking platform. (see Note 9)

8. To the blocking buffer 1:100 of anti-DNPH antibody is added and incubated for 1 h at room temperature on a rocking platform.

9. The primary antibody is then removed and the membrane washed three times for 5 min each with 50-mL wash blot.

10. The secondary antibody (Anti-Rabbit ALP-conjugated) is freshly prepared for each experiment as 1:3,000 in wash blot and the membrane is incubated on a rocking platform for 1 h.

11. The secondary antibody is discarded and the membrane is washed three times for 5 min each with wash blot.

12. After final wash the blot is developed using Sigma Fast tablet (see Note 10).

13. Normally, 10-30 min are necessary for color development. After color development, the developer is drained and the membrane is washed with tap water and dried between Kimwipes. An example of the results produced is shown in Fig. 11.2.

3.6. Image Analysis

1. SYPRO Ruby stained gels are scanned using a UV transilluminator (X = 470 nm, X = 618 nm).

2. The 2D oxyblots can be scanned with Adobe Photoshop on a Microtek Scanmaker 4900.

3. Oxyblots and 2D gel maps are matched with PDQuest image analysis software to determine the levels of specific protein carbonyls. The carbonyl immun ore activity of the oxyblot was normalized to the actual protein content as measured by the intensity of a protein stain such as SYPRO ruby.

4. The protein spots showing a significant increase in protein carbonyl levels are excised from the gel and in-gel-digested

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