20150420_bis transfection CNTN1-human(maxi Gisela)

Objectives

In order to know if the maxi-prep is (Origene) CNTN1-myc-DDK or (origene) CNTN1 without tags.

Material

• Coverslips
• 35mm cell culture plates
• Poly-D-lysine
• HEKs cells (Miquel)
• HEK Medium (FBS, Horse serum, pyruvate, pen-strep, glutamine)
• PFA 4%
• PBS
• Goat 5%
• Antibodies:
  • mouse-anti-myc
  • Rabbit-anti-CNTN1
  • mouse-anti-CNTN1
  • GAH (IgG) – 488
  • GAM-594
• Opti-MEM
• Lipofectamine 2000
• DNA:
  • CNTN1-human (maxi from 4ºC – Gisela), 0.4ug/ul

Methods

20.04.2015

1. Put 4-5 coverslips in a 35mm cell culture plate
2. Incubate them with poly-D-lysine 1:500 in PBS for an hour at 37ºC
3. Vacuum the poly-D-lysine
4. Wash 2 times with PBS and let the plates dry
5. Put 450.000 HEK cells in the plate o/n at 37ºC (Miquel)

21.04.2015

1. Prepare 4ul of lipofectamine 2000 in 125ul Opti-MEM
2. Prepare 3ug (7.5ul) of DNA in 125ul Opti-MEM
3. After 5 minutes put the lipofectamine solution in the DNA solution and incubate it at RT 1h
4. Aspirate off old medium from the plate and add new medium
5. Transfection: add the solution (250ul) to the cells and incubate it o/n

22.04.2015

1. Wash 2 times with PBS
2. Fix with PFA 4% 10min
3. Wash 2 times with PBS
4. Permeabilize with triton 0.3%
5. Wash 2 times with PBS
6. Block 1h with Goat5%
7. ICC
   1. Mouse-anti-CNTN 1/5000 | GAM-594
   2. Rabbit-anti-CNTN 1/120 | GAR594
   3. HVH1 1/40 | GAH-IgG-488
   4. Mouse-anti-myc | GAM-594
8. Wash 3 times with PBS
9. Mount with DAPI

Results

1. There are cells but there are not positive with this antibody
2. There are cells but there are not positive with this antibody
3. Patient has very positive marks in transfected cells
4. There are positive marks in transfected cells

Conclusion

•  Cells were transfected
• The plasmid was from CNTN1-mycDDK (Origene)
• The commercial CNTN1-antibodies don’t work how we want