My PBL Project

Question/Problem: What will be the molecular weight and charge of certain proteins if I run them through an electrified medium?

Hypothesis: If I test different proteins that have differing charges and weights, then certain proteins will become positive while others negative and speed of movement will differ.

Variables:

Independent- proteins

Dependent- The speed and direction

Controlled- Voltage, amount of protein. and environment

Materials

Electrophoresis Chamber

Agarose gel

Power Source

50x Concentrated Buffer

Gloves

Graduated Cylinder

Micro-pipette

Loading Tips

Electrophoresis dyes

  • Alazarin Red S Solution,

  • Malachite Green Solution

  • Orange G

  • Safranin O

  • m-Cresol Purple Indicator Solution

Procedure

Making Agarose Gel:

The first thing that you need to do is prepare the agarose gel that is needed to used because it provides a medium for the proteins to move through. First you need to put the dams to make sure no agarose flows out and keeps it secured. Then put a well comb to make wells that you will insert the proteins in the gel. Since the gel will be in a solid state already you must warm the solid to make it a liquid ( you may warm in the microwave or give the gel a hot water bath).Pour the agarose gel pour i halfway. Make sure you're wearing gloves because the agarose is an irritant to skin. Pour the gel . Wait around 20 minutes or until the gel becomes slightly opaque and hard.

Making the Buffer

After the the agarose gel has hardened insert it into the electrophoresis chamber and then it's time to make the buffer. If your buffer is concentrated then you will need to dilute it. The buffer that I was using was concentrated 50x so I put 2 ml of water and then added 98 ml of distilled water to make 100ml, Make as many needed to cover the gel considering every chamber has a different size.

Pipetting the proteins

The next step is going to be loading the proteins into the wells of the gel . Pick up the loading tip by pressing the micro-pipette button all the way down and then release. When inserting the protein press all the way down and making sure not to release before you go all the way into the gel before to not get air bubbles. Feel your way into the well and slowly press until you can see it has went in. It’s okay and normal if a portion of the sample floats to the surface because it will travel the proper direction anyway. Then connect the red to the positive and the black to the negative of the power supply. Turn it to 75 volts and watch it to see the charge and the molecular weight.

Data Table

Data Analysis

Alazarin Red S, Orange G, and m-Cerol Purple were all positive and Malachite Green and Safranin O are negative. Alazarin S is the fastest of the five proteins and m-Cerol is the slowest.

Conclusion:

All the proteins successfully ran through the electrophoresis chamber and .Alazarin Red S, Orange G, and m-Cerol Purple had positive results; two of the three of the positive were the fastest. All of the speeds directly correlated with their molecular weight. Some of the proteins never went to the end of the gel because of the weight.