Historical content from CDI L1 antibodies from Docollab
Summary
- Screening with LD401 PO with anti-FLAG beads
Incubate with primary antibody @ 4°C overnight
Anti-ORF2 mAb 1:1000 (clones 1-9)
Anti-ORF1 control (2mg/ml from Abmart) 1:2,500
Anti-FLAG control (4mg/ml from Sigma, M2) 1:1,000
- Screening with LD401, LD288 whole cell lysates
Probe with primary antibody overnight (Orf2 positive clones 1:2000 and anti-FLAG 1:2000 and anti-ORF1 1:5,000)
• CDI antibodies screening with LD401 PO with anti-FLAG beads
LD401 – ORF2-3xFlag
Scale: 500mg
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Anti-FLAG beads: 10ul per 100mg IP
Elution with 100ul of 1x LDS
8 % Acrylamide/Bis gel
- 1st Batch (supernatant) of antibodies from CDI 09/18/15
Incubation with primary antibody @ 4°C overnight
Anti-ORF2 supernatant 1:10 (clones 1-22)
Anti-ORF1 control (2mg/ml from Abmart) 1:5,000
Anti-FLAG control (4mg/ml from Sigma, M2) 1:2,000
(From 09/18/15 (Material: Elution of LD401 anti-FLAG PO)
https://www.docollab.com/project/779946374483208?folder=6265876214972416&item=779946374483208-1482577846826-34cd646b3097472697509298efa60fc9)
Strips 1-22: probed with cell culture supernatants from clones 1-22 (see below)
Strip 23 (ORF1): probed with anti-ORF1 antibody
Strip 24 (FLAG): probed with anti-FLAG antibody
- 2nd Batch (purified mAb) of antibodies from CDI 11/10/15
Incubate with primary antibody @ 4°C overnight
Anti-ORF2 mAb 1:1000 (clones 1-9)
Anti-ORF1 control (2mg/ml from Abmart) 1:2,500
Anti-FLAG control (4mg/ml from Sigma, M2) 1:1,000
11/10/15 (Material: Elution of LD401 anti-FLAG PO)
https://www.docollab.com/project/779946374483208?folder=6265876214972416&item=779946374483208-1482577846826-3b9f1c2243564f07b4737661949ffae9
Strips 1-9: probed with mAb from CDI (see below)
Strip 10 (ORF1): probed with anti-ORF1 antibody
Strip 11 (FLAG): probed with anti-FLAG antibody
• CDI antibodies screening with LD401, LD288 whole cell lysates
LD288 – ORF1-TEV-Flag (low expression, final grind, 1/6/2012)
LD401 – ORF2-3xFlag (LD401A, 2/15/14 harvested by John)
Scale: 100mg
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Triton X-100, 500mM NaCl (with protease inhibitors)
100mg of powder resuspended in 400ul of extraction buffer with protease inhibitors (sonication and extract clarification)
Load 5ul of reduced lysate per lane
Run 26-well 4-12% Bis-Tris gels for Western analysis
- 1st Batch (supernatant) of antibodies from CDI 09/25/15
(From 09/25/15 (Material:) https://www.docollab.com/project/779946374483208?folder=5943648491405312&item=779946374483208-1482579683310-03962bde80114dbf8cdacf8838462b86)
Cut 8-strips with Marker; LD401; LD288 on each one
Use 6 of them to probe Orf2 positive CDI clones: #7, 8, 11,13 and 19 and one for anti-FLAG positive control
Probe with primary antibody overnight (Orf2 positive clones 1:20 and anti-FLAG 1:1000)
Each strip includes Marker, LD401 whole cell lysate, LD288 whole cell lysate
- 2nd Batch (purified mAb) of antibodies from CDI 11/10/15
(From 11/12/15 (Material: LD401, LD288 whole cell lysate) https://www.docollab.com/project/779946374483208?folder=5734687997689856&item=779946374483208-1482577863163-0a65931bf1704ba3a4a68ef908e72304)
Probe with primary antibody overnight (Orf2 positive clones 1:2000 and anti-FLAG 1:2000 and anti-ORF1 1:5,000)
Cut strips with Marker; LD401; LD288 on each one
Use 6 of them on the other blot to probe mAb of anti-ORF1 clones: #5, 9, 10, 12, one for anti-ORF1 (from Abmart) and one for anti-FLAG positive control (Sigma M2)
Use 6 of them on one blot to probe mAb of anti-ORF2 clones: #7, 8, 11,13,19; and one for anti-FLAG positive control (Sigma M2)
Each strip includes Marker, LD401 whole cell lysate, LD288 whole cell lysate