13th week of SIP- Lab Techniques( Research)

Hi all, i think i miss out on my turn to blog one of those weeks.. so its ME again..
This time I m going to share with you a new assay I learnt: Co- Immunoprecipitation assay or in short, pull- down assay.

This assay works likes a protein- antibody reaction and with this assay, we can deduce whether are 2 proteins interacting with one another based on the Western Blot (WB) analysis which will be done after this.
For e.g: I wan to study if analog A binds to protein Z. normally, we will tag the analog A and protein Z with something so that we can detect them. Protein Z is tagged with the FLAG tag while HA tag for the TRX- analog and TRX proteins.
The analog A is attached to this protein scaffold (thioredoxine or TRX) to stabilize the structure.

I will have 3 reaction samples:
1. (-) Ctrl: Vector + Protein Z
2. (-) Ctrl: TRX + Protein Z
3. TRX- analog A + Protein Z
All this three DNA samples will be transfected into HeLa cells and then, we will lyse the Hela Cells to get the proteins translated.

After we lyse the cells and the cell Lysate are ready, we will save an aliquot mixed with protein dye without reducing agent for WB. The remaining will be topped up to 600- 1000ul with the lysis buffer.

Next, we will utilize beads. There are 2 types of beads: Protein G and A. however, I m using only the protein G. the protein G is attached to a solid matrix like agarose or sepharose and can bind to Ab. the beads will help to pulldown ab- protein complexes and when eluted, the complexes will detach from the beads and ready to load for WB analysis. (NB: Beads are hard to handle!!! So small and tiny and must be very careful not to suck them out in each step!)
Procedures:
Firstly, the beads will be pre-washed .
The lysate are then added to our pre-washed beads, and rotated at 4 °C for 1 hr. This step is called pre- clearing. We want to clear away those proteins in the lysate that will have an affinity to bind to the beads which can give non- specific bands under WB.
After that, the beads are spun down and the supernatant are transferred to another newly washed beads coupled with the Ab and rotated overnight at 4°C.
The next day, the beads are washed 5 times with wash buffer and eluted with 1 X TBS and protein dye. Proteins complexes that are bound together to the beads will therefore detach from the beads and can be loaded for WB analysis.

This assay often gives messy WB results. If we use a rabbit Ab for pulldown, we must use the mouse Ab for WB primary Ab and vice versa. The rabbit Ab will always give more background signal from WB than the mouse. Normally, I will also load the Lysate besides my pulldown in my WB. This allows me to compare and also troubleshoot whether my proteins are expressed in the Lysate or not. If my proteins expression is not detected in my lysate but I have a signal in my pulldown, it means that there might be something wrong in the experiment that can react to give a signal even my proteins are not around.
Any interaction can be seen based on the WB results and checked accordingly with the control to confirm that the experiment is working well and not due to any errors.
The success of pull down and protein expression will also depend upon the cell density for transfection and amount of DNA introduced into the cells.
So that’s all for the beads session. Feel free to ask me anything.
Hope you all are enjoying yourselves!!!

Ai Tee
TG 01
0503160D