Please explain what the experiment is showing me. Discussion answer 2-3 paragrap
ID: 264456 • Letter: P
Question
Please explain what the experiment is showing me. Discussion answer 2-3 paragraphs long.
Western Blotting Protocol
Week 1: Separate proteins in the samples by SDS-PAGE and transfer the proteins onto a biological membrane, nitrocellulose (NC) membrane. When we separate the proteins in the gel, we will divide the gel in half for Coomassie Stain and protein transfer. We will stain half of the gel with Coomassie Brilliant dye for 30min and I will destain the gel for a few hours after class. We will take the other half and transfer proteins onto the NC membrane using the iBlot instrument. When finished, we will stain the membrane with ponceu stain if time permits. It is an acidic dye that stains proteins red so that we can check the efficiency of the protein transfer. After observing the proteins on the NC membrane, we will leave the membrane in blocking solution.
Methods:
1. Obtain gel box (apparatus), a power supply, and a gel (you will share a gel box and power supply with another group), and samples labeled 1, 2, 3, and 4 and a protein standard ladder.
2. Peel off the gel sealer at the bottom of the gel if there is any.
3. Assemble the gel box with your gel and the other group’s gel (call me over) and pour running buffer into the box. Make sure the wells are submerged.
4. Load 10 ?l of protein ladder and samples in the order. Notice there are 10 wells, skip well 6.
5. Connect to the power supply and run the gel (I have to do this so call me when ready)
6. When the front blue dye (bromophenol blue) reaches more than 2/3rdof the gel, stop the power supply and take out the gel. Pour the running buffer back into the graduated cylinder and rinse the gel box thoroughly with water before bring it back to the cart.
7. Open the gel edges by gel knife and cut the gel in lane 6 vertically. Cut out the gel parts that do not have any proteins.
8. The side that has no protein ladder: Drop it into Coomassie Brilliant dye (use petri dishes), and the gel side that has protein ladder, bring it to iBlot for protein transfer.
9. Once the transfer is completed, cut out the membrane smallest possible that fit into the column I have. Check with me.
10. Pour ponceu stain over (petri dish) and rock it for 1 min. Pour back the ponceu stain into the bottle and wash the membrane with diH2O several times until you see red/pink bands (you can drain the waste into sink). Take a photo. This is your result. Put the membrane into the column I have and we will add blocking solution until we meet next week.
11. Pour the Coomassie Brilliant dye back into the bottle and wash the gel with diH2O several times (waste goes into waste bottle). You should not see any distinct protein bands because the dye stains background as well. This is your another result. Bring back your gel with your group’s name on the petri dishes.
Discussion: You need to discuss about your samples and what you saw in the gel as well as on the membrane. Also discuss the dyes used.
Week 2: This is a development step where we can observe p65 but not the other proteins. You have visually checked total proteins in the gel as well as on the membrane last week.
Methods:
1. Observe destained gel from last week and this is one of you results today. Be able to label the lanes and explain what you observed.
**This is an exam week so during your exam time, I would be proceeding the methods for you.
2. The blocked membrane should have been incubated on a rocker at 4C with primary Ab (Rabbit anti-p65 that is diluted in Ab solution) overnight
3. Washed the membrane for 3X with 1X TBST (wash buffer) on a rocker 5 min each
4. Incubate the membrane with secondary Ab (Goat Anti-Rabbit Ig conjugated with HRP) for 1 hour on a rocker at room temp
5. Wash the membrane 3X with 1X TBST (wash buffer) on a rocker 5 min each
6. Take your group’s membrane and add small amount of substrate (just enough to cover the membrane). Observe the color precipitation. If the background is changing color, discard the substrate into the sink. Take a photo. This is your second result.
Discussion: You should be able to discuss what you observed from the destained gel as well as what was developed on the membrane when you used anti-p65 antibody. Does the result support what you have expected (you should know what your samples are)? How so?
Explanation / Answer
Please explain what the experiment is showing me. Discussion answer 2-3 paragrap