Western blot is a common technique used in molecular biology aids the analysis of protein. One of the important steps involved is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) during which proteins in the sample of interest will be separated by a hydrogel matrix into groups/clusters/bands according to their molecular weight. Thus, the properties of the hydrogel is part of the crucial factors determining the success of the experiment.
Although the protocol for making such hydrogel (SDS-PA gel) is very well established and widely available on the web (e.g. youtube), the protocol still has space for improvement. The aim of this experiment is to optimize the efficiency of making a SDS-PA gel. Since SDS-PA gel formed by the principle of radical polymerization in which ammonium persulfate (APS) serves as the radical initiated by tetramethyl ethylene diamine (TEMED) while inspiring by the commercially available APS/TEMED tablets, a hypothesis of the feasibility of making aliquots from a mixture of APS and TEMED is raised.
Step 1: Materials Required
1) SDS-PA gel setting aparatus (in this case, Bio-Rad systems are used).
2) Reagents and Chemicals (as shown in the figure showing the recipe of 4% stacking gels).
3) Appropriate liquid deliverer with precision to the microlitre scale (in this case, eppendorf pipettes are used).
4) Appropriate liquid containers.
1) acrylamide is a toxin and a potential occupational carcinogen.
2) ammounium persulfate (APS) is a potential asthma inducing agent.
Step 2: Preparations (Part A)
2) Adjust the pipettes to appropriate volume.
3) Prepare the chemicals/solutions neccessary for making the 4% stacking gel if not have one.
4) Setup the glass plates (gel tank) as shown below:
* As both resolving gel and stacking gel are consisted of the same constituent (except the concentration and pH of the Tris buffer), stacking gel is chosen for demonstration driven by the following logic: 1) a less concentrated gel solution is less likely to form solid gel, thus if this stacking gel works then the resolving gel is expected to work under the assumption that there is no significant effect contributed by the differences in concentration and pH of the Tris buffer solution; and 2) lesser resources are needed.
Step 3: Preparations (Part B)
1) In addition to the APS/TEMED mixture aliquot, also prepare a pure APS aliquot (i.e. without TEMED).
2) After aliquoting, put the 2 tubes into -80 degree celcius ultralow freezer.
3) While the aliquots are freezing, prepare the 30% SDS-PA gel master mix (Tris buffer, sodium dodecyl sulfate, and 30% acrylamide/bisacrylamide mixture).
4) When the aliquots are frozen, take one APS/TEMED aliquot and two APS aliquots out of the freezer and start thawing.
* Since the SDS-PA gel shall start solidifying when the acrylamide/bisacrylamide units encountered the APS radical (initiated by TEMED), both APS and TEMED are usually the last to added.
1) Body temperature (e.g. holding the tubes by fingers) will thaw the frozen tubes faster.
2) Glass plates with different spacers may require different volumes of the gel solution to fill to the same height.
3) For 1 mm spacer glass plates (hold up to 5.5mL volume), 5mL is enough for the resolving gel while 1mL is enough for the stacking gel.
Step 4: Settings
Once all three aliquots are thawed, take one of the two APS only aliquots to the 4 degree celcius freezer and store for around 12 hours (when APS is expected to expire).
While the APS aliquot is freezing for 12 hours, the master mix of 30% acrylamide/bisacrylamide gel solution shall now be distributed into three 1.5mL microcentrifuge tubes (1mL solution each) labeled A, B and C.
For tube A, 10ul APS is added followed by 1ul TEMED.
For tube B, 11ul APS/TEMED mixture is added.
For tube C, 10ul 12-hour-incubated APS is added followed by 1ul TEMED.
Step 5: Loading
1) All completely mixed solutions are then vortexed (increase the saturation of the solution) and spin down (reunite all scattered droplets) before transfering to their corresponding gel tank.
2) After transfering the mixed gel solutions to the gel tank, propan-2-ol (i.e. isopropanol) is added on the top of the gel layer to prevent air reaching the gel solution (which are expected to inhibit the gel formation).
Step 6: Completed and Wait
Now, take a break with a timer. Usually the SDS-PA gel will solidify around 30 minutes but there are rumors that if left at room temperature for overnight the effect will be better.
Step 7: Results
Tube A and Tube C formed gel successfully while tube B remains in liquid form.
Step 8: Summary
These results suggested that:
1) SDS-PA gel can only be made successfully when APS and TEMED are separately added to the gel solution.
2) Aqueous APS lasts longer than expected (even after 12 hours).
3) APS/TEMED tablet may not manufacture by simply adding the two reagents together (at least not in solution).
The Chinese University of Hong Kong
Paper Brush Pot (2009)