(Difference between revisions)
Revision as of 22:08, 17 September 2013
Laser light path for Odyssey Licor Scanner. Image from Odyssey handbook.
Part 1: Cell Transfection
Today you will transfect cells with Lipofectamine LTX, a souped up version of the lipofection reagent that was used in Mod1. These cells will be used on M2D7 for your high throughput screen!
All manipulations are to be done with sterile technique in the TC facility.
Timing is important for this experiment, so calculate all dilutions and be sure of all manipulations before you begin.
You will set up four separate transfections; two experimental conditions and two controls.
|| [DNA] stock
|| Volume DNA
|| Volume OptiMEM
| RLuc-SH2 + EGFR-citrine
|| X μg/μL
|| 1.8 μg + 18 μg
|| X μg/ul
|| 1.8 μg
| RLuc + EGFR-citrine
|| X μg/μL
|| 1.8 μg + 18 μg
|| X μg/μL
|| 1.8 μg
|| 250 μL
- Obtain a 3 mL aliquot of OptiMEM from the center bench of the TC room.
- Calculate the amount of DNA you need to add for each transfection reaction.
- RLuc plasmid (empty psiCheck2 plasmid) = 1.0 μg/&mu:L
- EGFR-citrine = 1.2 μg/μL.
- RLuc-Grb2-SH2 plasmid = 0.8 μg/μL.
- RLuc-Shc3-SH2 plasmid = 0.6 μg/μL.
- RLuc-PLCg-SH2 plasmid = 0.9 μg/μL.
- Obtain one aliquot of Lipofectamine LTX from the center bench. There is 36 μL of LTX in each aliqout -- you only need one for today. Add enough OptiMEM to bring the total volume to 1000 μL in your tube.
- Vortex the OptiMEM/LTX mixture and incubate in your hood for 5-10 min.
- Meanwhile, label 4 eppendorf tubes to hold your DNA + OptiMEM: SH2/EGFR, SH2 only, RLuc/EGFR, RLuc only.
- Add 250 μL of OptiMEM to each of the eppendorf tubes you just labeled.
- Add the calculated volume of plasmid DNA to the appropriate tube.
- Add 250 μL of the OptiMEM/LTX mixture to each of your eppendorf tubes containing plasmid DNA. Mix by flicking the eppendorf tube with your finger. Incubate in your hood for 20 min.
- In the meantime, obtain a 12 mL aliquot of warm OptiMEM from the water bath.
- Take 4, 60 mm tissue culture dishes of CHO-K1 cells from the tissue culture incubator and change the medium to 2.5 mL of OptiMEM.
- Label the top of each dish with the transfection condition being used and your TEAM COLOR and put the plates back in the incubator until your transfection complexes are done incubating.
- Transfection: Using a P1000 pipetman, add 500 μL of transfection complexes drop wise while swirling the dish. Return the dishes to the incubator.
- Done! In 48 hrs your systems biology workhorse system will be ready to assay.
The Lipofectamine LTX / DNA complexes will incubate with your cells in OptiMEM media for 4 hours. After that, the teaching staff will remove the transfection reagents and add antibiotic free media to the cells. Tomorrow, the antibiotic free media will be replaced with normal growth medium.
Part 2.1: Day Two of Western Blot Analysis -- Secondary Antibody
Since you were last in lab, the teaching staff blocked your nitrocellulose membranes with Odyssey Blocking Buffer (OBB) -- a non-mammalian serum based buffer that is proprietary to Licor, Inc -- and then incubated the membrane with primary antibody at 4C on a shaker. The membranes were cut at the 50 kDa molecular weight marker so that we can evaluate GAPDH expression on the lower half and EGFR and p-EGFR on the top half. Today we will use infrared (IR) secondary antibodies and scan the Western blots using a specially constructed microscope located in the Lauffenburger lab to determine the phosphorylation of EGFR in response to your experimental conditions last time.
- Using a 500 mL glass bottle and the 20x TBS stock on the front bench, make a 500 mL 1x TBS stock using DI water from the lab sink.
- Now add enough Tween 20 to make a 0.1% solution (TBS-T). Tween-20 is located on the front bench. Shake the bottle well to mix.
- Obtain your blots from the front bench. Pour off the antibody solutions into the sink and add enough TBS-T to cover your membranes -- between 25-50 mL should work, but you don't need to measure this out. Keep in mind that the washing steps work by dilution, so it is a balance between adding enough to create a sink for the primary antibody, but not so much that you make a huge mess on the shaker!
- Shake your container for 10 min.
- Repeat for a total of 3 washes. Note: the time and number of washes was determined previously and can depend on your primary antibody!
- During your washes, dilute the secondary antibodies in 10 mL of OBB. They are light sensitive so find them on the front bench and then wrap your 15 mL conical in aluminum foil.
- For GAPDH (Rabbit) -- use the anti-Rabbit IR800 antibody at 1:15,000
- For EGFR (Goat) / p-EGFR (Rabbit) -- use the anti-Goat IR680 at 1:10,000 and anti-Rabbit IR800 at 1:10,000
- Why can we mix these antibodies?
- After the last wash, add your secondary antibody and place it on the dark shaker for 45 min.
- Pour off the secondary antibody and wash the membranes 3x, 7 min in TBS-T
- After the last wash, rinse the membranes 1x with 25 mL PBS. Pour off the PBS and keep in another 25mL of PBS until you can scan the membrane.
Part 2.2: Imaging the Western Blot
Sample placement for Western blot on Odyssey scanner. Image from Odyssey handbook
The Licor Odyssey scanner is a solid-state laser scanning microscope that concurrently images two wavelengths (700 and 800 nm), allowing for simultaneous detection of two (or more -- how?) antibodies.
The Odyssey scanner is located in the Lauffenburger lab in room 56-378. In groups of two you will go with a member of the teaching staff to scan your blots. The scanner has a variety of settings that control the resolution of the final image and the amount of light that is collected by the microscope objective. You will learn about those settings at the microscope. Be sure to note them in your lab notebook so that you may include them in your Methods section.
Scanning your Western blot is only the first step! You just did a lot of work to obtain what appears to be observational data. However, we can use densitometry to quantify the change in EGFR phosphorylation. Therefore, once you obtain your Western blot images -- which will be .tif files -- use ImageJ (a freeware for biological imaging from the NIH) to perform a densitometric analysis on your bands. A tutorial from NAVBO is attached here for instructions on performing densitometry. You will use this analysis in the FNT assignment for next time and it will help you to limber up your mind for the BRET analysis on M2D7.
Notes for Teaching Faculty
TA notes, mod 2