LaCava Research Wiki

Testing secondary fluorescent antibodies

Extraction buffer: 20mM HEPES, pH 7.4, 300 mM NaCl, 1% (v/v) Triton X-100 (with protease inhibitors)

Date: 07/28/20

Cell lines:

1. N2102Ep
2. NTERA

• Weigh out 2x 100mg of each cell powder, add 400ul of extraction buffer to each tube (1:4 w/v), vortex to mix. (Only small amount of cell was used for this experiment. Probably even 5 mg could be enough)
• New sonicator: Sonicate 5 x 2 sec at 2 Amp, power between 22J to 29J.
• Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
• Save 20ul of the input from each 100mg extract for Western (to test 2nd antibodies)
• Measure total protein concentration of saved cell extract by Bradford Assay.N2102Ep: 8.94ug/ul; NTERA: 6.58ug/ul
• Load 25ug of cell extract for Western on a 15-well Bis-Tris gel
• Add DTT to 50mM and LDS(1.1X) to cell extract and heat @70 °C for 10’
• Run 4-12% Bis-Tris gel to analyze the cell extract

1. Marker_5ul
2. N2102Ep_cell extract _10ul
3. NTERA_cell extract _10ul
4. BSA_5ul
5. Marker_5ul
6. N2102Ep_cell extract _10ul
7. NTERA_cell extract _10ul
8. BSA_5ul
9. Marker_5ul
10. N2102Ep_cell extract _10ul
11. NTERA_cell extract _10ul
12. BSA_5ul
13. Marker_5ul
14. N2102Ep_cell extract _10ul
15. NTERA_cell extract _10ul

• Wet transfer, 70V for 1.5 hours
• Cut the members into 4 strips, before the 2nd, 3rd and 4th marker
• Each strip will have 3 lanes

1. Marker
2. N2102EP
3. NTERA2

• Block two of the strip with TBST/5% milk at RT for 1-2hrs and other two with PBS+ 5%BSA
• Incubate with the primary antibody overnight
• 3 quick rinse with TBST
• Wash membrane with TBST, 3 x 10’
• Incubate with the 2nd antibody at RT for 1hr
• 3 quick rinse with TBST
• Wash membrane with TBST, 3 x 10’

We only incubate two of the strips (blocked with 5% BSA); one with BTUB (secondary 532nm, green) and the other ORF1 (secondary 637 nm, red).

Scan your membrane with QuantLAS4000. Make sure your color/channel lens is installed, physically change the light and calibrate using the specific plate. Finally choose the related filter through the software.

Result:

• BTUB antibody testing
• ORF1 antibody testing