10-24-19 – DC matrigel vs PC batch 2 comparison for immunoprecipitations with LaORF1 and mouse anti-ORF1 beads + MT302 vs N2102Ep test
Date: 10/24/19
Cell lines:
PA-1 PC Batch 2 (from Maria)
PA-1 DC (with matrigel, batch from July 2018)
N2102Ep
MT302 Transient
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl. Add protease inhibitors in extraction buffer 1:100; Add RNasin 1:250 in extraction buffer and 1:1000 in wash buffer
Scale: 25mg twice from PA-1 PC, 50 mg twice from PA-1 DC; 100 mg twice from PA-1 DC; 50 mg from N2102Ep, 25 mg from MT302.
Weight 25 mg/50 mg/100 mg and add 100 ul/200 ul/400 ul of extraction buffer to each tube;
Save 10 ul from N2102Ep and from MT302
Sonicate @ 2Amp for 3x 2sec – For 25 mg of PA-1 PC
Sonicate @ 2Amp for 5x 2sec – For 50 mg of PA-1 DC and
Sonicate @ 2Amp for 5x 2sec – twice – For 100 mg of PA-1 DC
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Set up the following IPs
1) PA-1 PC anti-ORF1 beads (25 mg – 5 ul of beads)
2) PA-1 PC LaORF1 XL+ beads (25 mg – 5 ul of beads)
3) PA-1 DC (50 mg) anti-ORF1 beads (50 mg – 5 ul of beads)
4) PA-1 DC (50 mg) LaORF1 XL+ beads (50 mg – 5 ul of beads)
5) PA-1 DC (100 mg) anti-ORF1 beads (100 mg – 10 ul of beads)
6) PA-1 DC (100 mg) LaORF1 XL+ beads (100 mg – 10 ul of beads)
7) N2102Ep (50 mg) anti-ORF1 beads (50 mg – 10 ul beads) (used double the amount of beads we used for MT302, because it is how we did with PA-1)
8) MT302 (25 mg) anti-ORF1 beads (25 mg – 2.5 ul of beads)
Total: 32.5 ul of beads mouse anti-ORF2 beads; 20 ul of LaORF1 beads
IP @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer by gentle pipetting
Switch beads to fresh tubes after the 2nd wash step
Wash a 3rd time and elute in 10 ul of 2% SDS 40 mM Tris pH 8 at 70ºC for 5
minutes (I did the elution in 10 ul but I load everything – I should have used 2.5 ul to keep the ratio, but this is nothing and changing the volume shouldn’t hurt)
Western blot
Run a 4-12% Bis-Tris gel to analyze the elutions. 10 well gel Load 5 ul of each elution. Add 4 XLDS and DTT to 50 mM and heat 70 ºC for 10 min.
200 V 40 min
1) Marker
2) PA-1 PC anti-ORF1 beads 10 ul - 5 ul 4XLDS, 1 ul DTT (stock 1M), 4 ul H20
3) PA-1 DC (50 mg) anti-ORF1 beads 10 ul - 5 ul 4XLDS, 1 ul DTT (stock 1M), 4 ul H20
4) PA-1 DC (100 mg) anti-ORF1 beads 10 ul - 5 ul 4XLDS, 1 ul DTT (stock 1M), 4 ul H20
5) PA-1 PC LaORF1 XL+ beads 10 ul - 5 ul 4XLDS, 1 ul DTT (stock 1M), 4 ul H20
6) PA-1 DC (50 mg) LaORF1 XL+ beads 10 ul - 5 ul 4XLDS, 1 ul DTT (stock 1M), 4 ul H20
7) MT302 (25 mg) anti-ORF1 beads 1 ul - 5 ul 4XLDS, 1 ul DTT (stock 1M), 13 ul H20
8) Marker
9) N2102Ep (50 mg) anti-ORF1 beads 10 ul - 5 ul 4XLDS, 1 ul DTT (stock 1M), 4 ul H20
10) MT302 supernatant (prepared the whole 10 ul, up to 20 ul - loaded 4 ul – equivalent to 25 ug)
11) N2102Ep supernatant (prepared the whole 10 ul, up to 20 ul - loaded 4 ul – equivalent to 25 ug)
Primary antibody
- Mouse anti-ORF1 Abmart 4H1 (2mg/ml) - 1:5000 o/n @ 4ºC
3x 15 min washes wih TBS-T
Secondary antibody
- Anti mouse-HRP - 1:10000 RT 1h 3x 15 min washes wih TBS-T
