WEEK THREE
- MONDAY 10/7/17 -
In the morning, I wrote up the western blot results from last week. The Odyssey scanner is very useful as it generates a digital image quite quickly. The images can be edited and saved both in colour and grayscale.
As there are 8 antibodies, I will be testing two at a time along with the rabbit FLAG antibody on one membrane. The results with the rabbit FLAG were reassuring as the strong bands confirmed the transfections had worked. Tetraspanins usually produce a characteristic band at approximately 30kDa, within the boxed region. However, it was a disappointing result, as there were no bands present - suggesting that the Tspan14 antibodies can't be used in western blotting. The results for mT14 are as expected, as the FACS data already confirmed that the Tspan14 mAbs don't recognise mouse Tspan14.
EXPECTED RESULT:
The empty vector (EV) was as expected, because the antibodies don't recognise endogenous Tspan14. I would have expected a band for hTspan14 with the two antibodies.
EXPECTED RESULT:
The empty vector (EV) was as expected, because the antibodies don't recognise endogenous Tspan14. I would have expected a band for hTspan14 with the two antibodies.
After that I checked the hybridomas, which were all very dense and dead. I spent two hours harvesting the tissue culture supernatant into falcon tubes and 2 ml aliquots for freezing.
In the afternoon, I checked my cells which were now confluent, counted the cells with a haemocytometer and and seeded two plates for transfection. Jonathon (a Masters student) helped me locate my cells under the microscope (turns out I was being crazy and looking at the empty side of the haemocytometer 😂). I also had enough cells to do another 1:5 split to keep the cells growing.
- TUESDAY 11/7/17 -
Today, I transfected the two plates with a human Tspan14 construct and an empty vector, which will be used in the FACS experiment tomorrow to test the new tissue culture supernatant antibodies compared with the old antibodies.
- WEDNESDAY 12/7/17 -
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| So many tubes!!! 😱 |
SUMMARY OF RESULTS
As expected the antibodies didn't recognise endogenous Tspan14 on the mock transfected cells.
Below are the results with hTspan14 transfected cells. There was very little difference between the shift peaks produced by the old and new antibodies.The only strange results was with 2H5, where both old and new TC supernatants didn't seem to recognise Tspan14 on the hTspan14 transfected cells either.
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| 2H5 being dodgy :( |
- THURSDAY 13/7/17 -
Today I decided to be brave and ran two gels at the same time to finish the western blot experiment with the remaining six antibodies: 2H5, 3G3, 6E5, 9D5, 9G1, 10F3 (using the new TC supernatants) and rabbit FLAG as the control. I immediately regretted it when Connie reminded me of how many washes I'll have to carry out tomorrow 😂 This time I did a 1:5 dilution of the antibodies rather than 1:10.
Whilst waiting during the membrane transfer step, I checked on my cells to see they were confluent. I did a 1:20 split so that they'll be ready for experiments next week.
After discussing the FACS data with Connie, she suggested we do another FACS experiment using APC for the secondary staining to see if the antibodies recognise endogenous Tspan14 (we're still being very hopeful!)
- FRIDAY 14/7/17 -
After washing the membranes, they were incubated with the secondary antibody (mouse 800) solution. On one of the membranes I also stained it with rabbit 680. These two antibodies can be visualised simultaneously in two different channels (red and green).
Expected result:
- Strong band with hTspan14 as the antibodies recognise Tspan14 on transfected cells in FACS experiments
- No band for any of the eight antibodies with the EV because they don't recognise endogenous levels.
- No band with mTspan14 beecause Tspan14 mAbs don't recongise mouse Tspan14.
RESULTS:
Again, the blots were disappointing as there was no band for the antibodies with hTspan14.
The strong bands with the rabbit FLAG confirms that the transfections were successful.
There was a strange patch at the bottom of the 3G3 membrane - I have no idea why! :P
Very nice data, Hanh!
ReplyDeleteIn addition to testing other cell lines, I think it's also worth trying APC staining again with the other 7 antibodies, to see if we can detect endogenous Tspan14, since we only did 2H5 in our first APC trial, which seemed to be the weakest one from the first two FACS experiments, and now seemed to have somehow gone unstable and not recognising even overexpressed hTspan14.
It's also nice to know that you have also seen reduced Tspan15 levels in cells overexpressing Tspan14 in your recent two experiments - it agrees with my previous data when I tested Tspan15 levels on HEKs stably overexpressing Tspan14. This is most likely because of the limited amount of ADAM10 - most of them have paired up with the overexpressed Tspan14, with very little ADAM10 left to pair up with Tspan14!
I also wonder if we would see a change in Tspan14 levels in Tspan15 KO cells... but it's a shame we can't see endogenous Tspan14 levels... yet! Let's continue being hopeful :D
I look forward to reading about your Western blot results - have fun with the tens and thousands of washes tomorrow :P
Connie
Yes - that sounds like a good plan! It is a real shame, but I'm still optimistic... maybe we'll see a tiny positive shift with APC :)
DeleteHaha - I'll be having a lot of fun with the washes today. I'm slowly regretting running all 6 antibodies at the same time now 😂