Protein separation

Carefully place the gels from 1 team in the gel electrophoresis holder (note the orientation – opening of slots to the inside) and pour electrophoresis buffer:

• All systems: from the inside to the top of the long plate. Do this first, then wait a minute to see if there is a leak anywhere. When the liquid level remains the same (completely full), you can continue.
• A gel system for 2 gels: on the outside up to 1 cm above the bottom of the gels.
• A gel system for 4 gels: on the outside to the indicated line for 2 or 4 gels, depending on how many gels are put in.

 Start the electrophoresis run – check for bubbles at the bottom.

• 80V, +/- 15 min, until samples reach the running gel.
• Increase the voltage to 200V for approx. 20-30 minutes.

Stop the run when the blue colored bands reach the end of the gel. First stop the program and unplug the appliance, only then remove the gels!

Note: Proceed immediately with the blotting. If you have to wait for an assistant,  keep your gel running on the lowest voltage (20V), so your proteins stay separated.

Bring back used stuff back to the assistent and refill stock.

Hand back to assistent:

• Samples (stocks)
• Protein marker
• Sample Buffer

Refill bottle with elektroforese buffer.

Why is it important that there are no system leaks in the gel electrophoresis system?

If in need of more explanation regarding the principle of SDS-PAGE, watch the “kennisclip” in the labbuddy environment under “gel gieten”.

1. A) The gel will not run correctly because the current is not forced through the gel but takes a ‘shortcut’ through the leak
   • Correct! The gel electrophoresis buffer has the purpose of guiding the passage of electricity between the anode and the cathode. With a system leak too much electrophoresis buffer will run into the outer tank leaving the top of the gel uncovered (see answer A). As a consequence, the current is not forced through the gel but takes a ‘shortcut’ through the leak, as it will seek the shortest way through a current bearing liquid.
2. B) The gel will not run correctly because too much electrophoresis buffer will run into the outer tank leaving the top of the gel uncovered
   • Correct! If the top of the gel runs dry no current will pass through the gel anymore and thus the proteins will not migrate.
3. C) It does not matter that there is a leak, the gel will run just fine
   • Incorrect. It does have an effect.
4. D) None of the above
   • Incorrect.

Here you see four pictures of gels inserted into the holder. Which is correct?

1. • 
     Incorrect. In this picture only one gel is placed in the holder leaving the other side of the inner chamber open.
   • 
     Incorrect. In this picture the gels are put into the holder too high resulting in a gap underneath the gels.
   • 
     Correct. This is the only picture in which the gels are properly installed. The rubber caskets can properly seal off the inner chamber and the small glass plate is oriented inward making it possible for the current to flow from the inner chamber- through the gel- to the outer chamber.
   • 
     Incorrect. Here the gels are placed with the small glass plate facing outward making it impossible for the electrophoresis to run.

Here you see four pictures of a gel electrophoresis at different times. Which band height correlates with the correct time to stop the electrophoresis?

Knowledge of protein marker and expected band sizes is expected.

• 
  Incorrect. The gel has not yet run long enough.
• 
  Correct! This is the right timing. The gel has run long enough to make enough distinction between the different MW proteins.
• 
  Incorrect. The gel has not yet run long enough.
• 
  Incorrect. The gel has run for too long. You can no longer reliably measure protein size because the front has run off the gel. Also proteins may have run off the gel.

Wat is juist met betrekking tot de bandjes op de gel na het runnen?

1. De bandjes die het verst beneden zijn, corresponderen met de kleinste eiwitten uit je samples.
   • Juist! Kleine eiwitten migreren sneller door de gel dan grote eiwitten. De onderste bandjes corresponderen met eiwitten die het snelst gemigreerd zijn, oftewel de kleinste eiwitten.
2. De bandjes die het verst beneden zijn, corresponderen met de grootste eiwitten uit je samples.
   • Ga nog eens na welke eiwitten het snelst door de gel migreren.

Wat is de functie van SDS in de elektroforesebuffer?

1. A) SDS behoudt de gedenatureerde vorm van de eiwitten
   • Incorrect. SDS heeft meedere functies.
2. B) SDS bindt aan eiwitten waardoor eiwitten een negatieve lading krijgen evenredig met hun grootte.
   • Incorrect. SDS heeft meedere functies.
3. C) Zowel A en B zijn waar.
   • Juist! SDS heeft zowel de functie om eiwitten in gedenatureerde vorm te houden als om de eiwitten een negatieve lading te geven. Hoe groter het eiwit is, hoe negatiever het geladen zal zijn. Deze negatieve lading zorgt ervoor dat eiwitten vanaf de negatieve elektrode (anode) naar de positieve elektrode (kathode) zullen gaan migreren.