The overall goal of this two-dimensional gel electrophoresis coupled with mass spectrometry procedure is to separate and identify human pituitary adenoma tissue proteome. This method can help answer key questions in the proteomes field. Such as high of proteome.
And discovery of protein situation, protein four, and protein spacious. The main advantage of this technique is that two dimensional gel electrophoresis is easily coupled with high sensitivity mass spectrometry to identify proteome, especially low abundance proteins. Protein situation, protein four, and protein spacious.
The implementations of this technique is tend toward PQ itery of animal therapy. Because of key protein for protein spacious or protein pattern is useful in patients diagnosis. We show a demonstration of this method is critical as the two dimensional gel electrophoresis steps are difficult to learn because it takes time consuming labor intensive and there are several experimental tricks.
To begin this procedure add 250 microliters of a previously prepared protein sample solution to each slot of a 13 centimeter IPG strip holder. Place an 18 centimeter IPG strip, gel side down, onto each protein sample solution taking care to avoid bubbles. Then add three to four milliliters of mineral oil to cover each IPG strip.
Next, assemble each IPG strip holder into an isoelectric focusing system with the pointed end on the back plate and the square end on the front plate. After rehydrating the IPG strips for 18 hours at room temperature, set the IEF parameters on the isoelectric focusing system with a total run time of 7.5 hours and 36, 875 volts hours. After IEF, take out each IPG strip from the isoelectric focusing system and remove the excess mineral oil with an insoluble paper towel.
Then wrap each strip in a sheet of plastic wrap and store at minus 80 degrees Celsius. To cast a 12%PAGE gel add double distilled water, 1.5 molar tris-hydrochloric buffer, 40%acrylamide, bis-acrylamide stock solution, 10%immodium persulfate, and timed to a beaker. Mix the gel solution gently, taking care to avoid bubbles.
Gently pour the gel solution into a multi-casting chamber up to the expected gel height of 19 centimeters. Immediately add about three milliliters of double distilled water to the multi-casting chamber to cover each resolving gel and allow each gel to polymerize for about one hour. This step is very important to prepare a high quality SDS-PAGE gel and the water must be immediately added on the top of each gel.
Prepare two liters of electrophoresis buffer then add the buffer to an electrophoresis separation unit buffer tank. After removing the focused IPG strips from the freezer, place them in a plastic vessel containing one milliliter of reducing equilibrium buffer and a trace of bromophenol blue and equilibrate them for 10 minutes with gentle rocking. After removing the reducing equilibrium buffer, add four milliliters of excalation equilibrium buffer and a trace of bromophenol blue to the IPG strips and equilibrate them for 10 minutes with gentle rocking.
During gel equilibrium, disassemble the multi-casting chamber and remove the prepared resolving gel cassette. Rinse the gel cassette three times with double distilled water, then remove access water from the gel cassette with an insoluble paper towel and place it in a gel stander. Following this, rinse the equilibrated IPG strips with electrophoresis buffer and remove excess liquid on each IPG strip surface with an insoluble paper towel.
Place an IPG strip onto the resolving SDS-PAGE gel, allowing the IPG strip's plastic side to contact the longer glass plate with the pointed end to the left. Now quickly add three to four milliliters of hot 1%agarose and SDS electrophoresis buffer to seal the IPG strip on the top of each SDS-PAGE gel and place the top side of the IPG strip down on the top of the shorter glass plate taking care to avoid bubbles. These steps are very important to seal the IPG strip to the SDS-PAGE gel.
The hot agarose solutions must be immediately added and the IPG strip must be immediately placed into the top of the SDS gel. After polymerization, insert the assembled gel cassette vertically between the plastic gaskets in a vertical electrophoresis system. Place the top of the gel with the IPG strip next to the cathode.
Next, adjust the level of electrophoresis buffer to immerse the gel cassette by adding the appropriate volume of buffer to the electrophoresis system. Connect and set the power supply in constant voltage mode and run at 200 volts for about 370 minutes while monitoring the dye. After the run is complete, take the gel cassette out of the electrophoresis system and gently remove the gel from the gel cassette, taking care to avoid tearing the gel.
Carefully transfer the gel to a tray containing 250 milliliters of fixation solution and gently shake the gel for 20 minutes. Following this, discard the solution and add 250 milliliters of 50%methanol to the tray. Then gently shake the gel for 10 minutes.
After shaking the gel in double distilled water, add 250 milliliters of sensitization buffer containing 0.02%of sodium sulfate. Gently shake the gel for one minute. After shaking the gel twice in double distilled water, add 250 milliliters of silver reaction solution and gently shake the gel for 20 minutes.
After shaking the gel in double distilled water, add 250 milliliters of development solution and gently shake until the desired staining is obtained, which is typically one to three minutes. Now add 250 milliliters of 5%acidic acid to terminate staining and gently shake the gel for 10 minutes. After shaking the gel in double distilled water, add 250 milliliters of 8.8%glycerol and store the gel at four degrees Celsius until analysis.
A reproducible and good two dimensional gel pattern of NFPA tissue proteome was obtained from gel electrophoresis with approximately 1, 200 protein spots detected in the two dimensional gel map that were mainly distributed within the area of pH four to eight and mass 15 to 150 kilodaltons. A total of 192 redundant proteins were identified from 141 gel spots out of the 337 analyzed gel spots in the MALDI-MS PMF analysis and no protein was identified in the remaining 196 spots. Staining with commassie blue yielded a reproducible and high quality two dimensional gel pattern of invasive NFPA tissue proteome and approximately 1, 100 protein spots detected in a two dimensional gel map that were mainly distributed within the range of pH four to eight and mass 15 to 150 kilodaltons.
An average of one protein per spot was identified with MALDI tof tof MS MS analysis. Where as many proteins in each corresponding spot were identified with LC-ESI-MS/MS analysis. This data clearly demonstrates that each two dimensional gel spot contains many proteins in the complex human cancer tissue proteome.
Once mastered this technique can be done in 32 hours for two dimensional gel electrophoresis if it is performed properly. While attempting this procedure, it is important to remember to follow the steps, especially according to the protocol. Following this procedure, other methods like stable labeling can be performed in order to answer additional questions like the codification of proteins in the proteome.
After its development, this technique paved the way for researchers in the field of protein mix to explore larger scale low abundance protein post translational modifications, protein specialization, protein spacious, and protein four in model conditions, patients, and organ systems. After watching this video you should have a good understanding of how to perform two dimensional gel electrophoresis coupled with mass spectrometry achieving good and reproducible two dimensional gel electrophoresis pattern and identify many proteins in each spot. Don't forget that working with SDS achreomy and TEMED can be extremely hazardous and precautions such as wearing plastic gloves, should always be taken while performing this procedure.
