WEEK TWO
- MONDAY 3/7/17 -
Dr Tomlinson gave me a plan of the experiments I could conduct this week and for when he is away at a conference and on holiday. The experiments I'll be conducting this week will be similar to last week's, but I will also be testing if the Tspan14 mAbs recognise mouse Tspan14 as well as human Tspan14 by flow cytometry and western blotting.
To prepare for tomorrow, I needed to seed plates for the transfection planned. However, upon checking the plates from last Friday, the HEK cells hadn't grown as well as expected, which was strange because it is quite a fast growing cell line. Usually when the cells have divided and are growing, the media will change to a orange/yellow colour. However this wasn't observed with my two plates. There weren't any signs of infection - however Connie suggested the cells might have started to die/ been contaminated... so I'm disappointed the cells didn't grow as well as I'd hoped. ðŸ˜ðŸ˜¢
We went ahead anyway to see if there were enough cells to carry out the experiment. I followed the protocol for splitting cells and then pipetted 10ul onto haemocytometer to count. There were not enough cells to be able to go ahead with FACS. FACS requires 3000-10,000 cells, but it is better to have more, usually between 250,000 - 500,000. We split a new plate of my cells to see if they will grow during the week.
I will have to wait for Connie to count her HEK cells and see if she can split three plates for the FACS experiments, as her cells were a lot more confluent.
- TUESDAY 4/7/17 -
WE HAVE CELLS!! 🎉
Connie counted her cells yesterday and had enough to split three plates for me to transfect. I took a look at her plates and they were 50-60% confluent, which is ideal for transfections. We also looked at my plate of cells and it was a nice surprise to see that the cells are in fact alive and growing!! 🎉🎉
I transfected the three plates of cells as follows:
a) pEF6
b) pEF6 FLAG-human Tspan14
c) pEF6 FLAG-mouse Tspan14
After the transfection, Connie helped me plan the FACS experiment for tomorrow and explained how we were going to harvest the lysate for western blotting.
Summary of results:
We then analysed the samples by FACS. The results were similar to the previous experiment, which confirmed that the Tspan14 mAbs don't recognise endogenous Tspan14. We also ran a sample stained with APC rather than FITC as the secondary stain. However the results still show that endogenous Tspan14 cannot be detected even with a brighter fluor. We also tested for interaction of the antibody with mTspan14 (mouse), however none of them recognised mTspan14, unlike some of the Tspan15 clones which also recognised the mouse protein. However, there isn't evidence from FACS to determine whether the transfection worked - so we will run a western blot tomorrow.
We still seemed to have a problem with weak signals when the samples are run on low. Nobody else in the lab has had this problem - so I've come to the conclusion that the FACS machine simply doesn't like me!
Update on my cells! :)
After collecting all the data, I checked my HEK cells to see if they were still growing. They appeared ~80% confluent so I did a 1:5 split.
- WEDNESDAY 5/7/17 -
My first full day of non-stop experimenting was exciting but also exhausting!
The day started off with a lab meeting and I presented the FACS data I'd analysed from last week.
 |
| Tspan14 Transfected Cell |
 |
| Mock Transfected Cells |
My data showed a positive shift in fluorescence with the Tspan15 antibody in the Tspan14 transfected cells. However Connie performed a similar experiment and didn't see this result, so we will repeat that today with another FACS experiment using 5D4.
FACS sample prep
We checked the 3 plates of transfected cells and decided they were ready for the FACS experiment. Having watched Dr Tomlinson lead the FACS experiment last week, Connie let me carry out the sample prep, whilst she watched. As we had enough cells left, we harvested the supernatant to do a western blot tomorrow.Summary of results:
We then analysed the samples by FACS. The results were similar to the previous experiment, which confirmed that the Tspan14 mAbs don't recognise endogenous Tspan14. We also ran a sample stained with APC rather than FITC as the secondary stain. However the results still show that endogenous Tspan14 cannot be detected even with a brighter fluor. We also tested for interaction of the antibody with mTspan14 (mouse), however none of them recognised mTspan14, unlike some of the Tspan15 clones which also recognised the mouse protein. However, there isn't evidence from FACS to determine whether the transfection worked - so we will run a western blot tomorrow.
We still seemed to have a problem with weak signals when the samples are run on low. Nobody else in the lab has had this problem - so I've come to the conclusion that the FACS machine simply doesn't like me!
Update on my cells! :)
After collecting all the data, I checked my HEK cells to see if they were still growing. They appeared ~80% confluent so I did a 1:5 split.
 |
The cells that we thought were dead,
are in fact growing and now confluent! 😊
|
- THURSDAY 06/7/16 -
 |
| Second time lucky!! |
This morning, Connie demonstrated how to make a gel and let me make my own. Everything was fine with the lower buffer. However, we forgot to check if the gel had set before pouring off the water - so ended up having to make another gel 😂
The first step in western blotting is SDS-PAGE to separate the proteins by size. Loading the samples into the almost invisible wells was definitely the trickiest part!
After running the gel, I assembled a membrane sandwich in the transfer cassette to transfer the proteins from the gel onto the membrane at 30V for 90min. The membrane was blocked in milk for an hour at room temp. to prevent non-specific binding of the antibodies. The membrane was then cut and stained with primary antibodies: 1A3, 1B6 and rabbit FLAG and incubated overnight.
The first step in western blotting is SDS-PAGE to separate the proteins by size. Loading the samples into the almost invisible wells was definitely the trickiest part!
After running the gel, I assembled a membrane sandwich in the transfer cassette to transfer the proteins from the gel onto the membrane at 30V for 90min. The membrane was blocked in milk for an hour at room temp. to prevent non-specific binding of the antibodies. The membrane was then cut and stained with primary antibodies: 1A3, 1B6 and rabbit FLAG and incubated overnight.
Analysis of FACS data
As seen last week, none of the antibodies detected endogenous Tspan14 - even with APC.
 |
| All 8 antibodies produced a positive shift in fluorescence with human Tspan14 transfected cells |
 |
| None of the antibodies recognised mouse Tspan14 |
 |
| A repeat with Tspan15 antibody - 5D4 did not produce a positive shift in fluorescence in Tspan14 transfected cells as last week. |
- FRIDAY 7/7/16 -
 |
| They're so tiny!!! |
 |
| Last washing step!!! :) |
After the talk, there was a lot of waiting around as I had to wash the membrane several times. After the washing step, the membranes were dried and stored in a box. Then there was more waiting around to do as a lot of people wanted to use the Odyssey scanner :(
I checked my cells again and they seem to be growing nicely. I did a 1:5 split into one plate so that hopefully, they'll be confluent on Monday.
Comments
Post a Comment