We decided to switch to try to capture endogenous ORF1 instead of endogenous/exogenous ORF2 from DC and PC. (Because we only were able to detect ORF1, not ORF2)
To do this, we were growing PA-1 PC and DC cells to prepare BBs:
07/06/18 - PA-1 PC culture for getting BBs
07/10/18 - PA-1 DC culture for getting BBs
Once we have the material, first of all we wanted to find the optimal IP conditions to get the best ORF1 amount possible.
07/23/18 - IP from PC and DC with different amounts of beads
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Hua titrated the mouse anti-ORF1 beads (09/24/18 - MT302 and PA-1 PC PO with anti-ORF1 beads) and LaORF1 beads (10/09/18- Anti-ORF1 IP with MT302 and PA-1 PC cells: titration of LaORF1 beads) for PA-1 IPs.
The amount of beads that gave the best ORF1 yield was 10 ul of beads per each 50 mg of PA-1 powder.
She also did anti-ORF1 IPs with PA-1 and MT302 material for MS to get preliminary results (02/27/19 - PA-1 and MT302 purification for MS).
The results looked promising (a potential new locus for ORF2 expression was found on PA-1), however, aditional controls were neccessary to confirm this.
Then we decided to repeat the experiment in a bigger scale (500 mg of powder instead of 100 mg, in order to get a more intense signal) using mouse anti-ORF1 beads for IP (07/24/19 - Capturing RNP with mouse anti-ORF1 beads from PA-1 PC - With anti-mIgG beads as a control).
- We had problems with the elution in 1/2 S-trap buffer at RT for 10 min, ORF1 didn't elute as observed in the Sypro (but the IP worked because the elution for Western was done in LDS and we got a signal). To solve this, we tested different elution buffers/temperatures for IPs:
08/05/19 - Anti-ORF1 IP with MT302 to test different elution buffers for S-Trap
The best condition after testing them, was the 2% SDS 40 mM Tris pH 8 at 70 ºC for 5 min.
- From the western blot we know that PA-1 has 10 times less ORF1 expression than MT302 and that to detect ORF1 from PA-1, both in sypro and western blot, we need to load the equivalent to 25 mg of powder. To get a band with the same intensity from MT302, we should load the equivalent to 2.5 mg of MT302 powder.
- From the gel stained with sypro, we observed that for 200 mg of PA-1 powder, using mouse anti-ORF1 beads for IP in PA-1, we were getting a giant inespecific band at 40 kDa. As this band could interfere with the MS and as the IPs with LaORF1 beads don't show this giant 40 kDa band, we decided to switch from using mouse anti-ORF1 (Abmart-4H1) to the LaORF1 beads. However it was neccessary to prepare more beads and test them.
Hua talked to Peter to prepare them. We tested them later.
07/31/19 - Conjugation of LaORF1-5, LaG94-10 and anti-FLAG Beads
- After this we tested the new LaORF1 and LaG94-10 beads. 08/05/19 - New LaORF1/LaG94-10 batch testing.
- However we were getting an intense 15 kDa signal equivalent to the light chain of the nanobody. Thus we wondered if the XL of the beads with the nanobody would give us a less intense 15 kDa band. Then, Hua did the crosslinking of an aliquot of the nanobody and I tested them.
08/05/19 - Orf2 (MT302) PO testing LaORF1 beads with or without XL
- Once we had the beads ready and we knew which elution to use, we proceed with 08/09/19 - Capturing RNP with lama nanobodies LaORF1 beads from PA-1 PC - With LaG94-10 beads as a control
We captured ORF1, both using mouse monoclonal anti-ORF1 (Abmart 4H1) and lama nanobody anti-ORF1 (LaORF1) coupled beads and give the samples (prepared by S-Trap) to Kelly for MS. Once we got the data, Mehrnoosh analized it generating a report.
Report file: https://docs.google.com/document/d/1b4Nuzp5UYxpWPOYoJK0yIyHZPzMnBxE3NlklVNa1x4M/edit?usp=sharing
Data files:
https://drive.google.com/drive/folders/1dsV95_nloMkhgKH-5EvZj5MsW6svBER8?usp=sharing
After reviewing the data, we decided to proceed with the DC set of samples.
We had different PA-1 DC powder:
- Powder I grew last year (I grew it with matrigel as a matrix)
- Powder from the PA-1 DC BBs I grew in Spain this year (2019) before I came for the 6 months stay
We didn't know how much PA-1 DC material would we need to weight to get an ORF1 yield similar to the yield obtained from PA-1 PC. Due to this, we decided to use the old PA-1 DC powder (with matrigel) to do a pre-check and use the new powder for the the MS experiment. After checking some previous data and doing some numbers/comparisons, we concluded Why PA-1 DC with matrigel has half the amount of PA-1 DC without matrigel - Rationale of the pre-check assay, so we finally planned a pre-check experiment based on this (considering that PA-1 DC with matrigel had half the amount of ORF1 than the PA-1 DC withtout matrigel).
10-24-19 – DC matrigel vs PC batch 2 comparison for immunoprecipitations with LaORF1 and mouse anti-ORF1 beads + MT302 vs N2102Ep test
The result from the test showed that the best for PA-1 DC, was to use the same conditions we had been using for PA-1 PC. PA-1 DC without matrigel should give a similar ORF1 yield to the PA-1 PC. Thus, we planned to 10/30/19 - Capturing RNP with mORF1 beads from PA-1 DC - With mIgG beads as a control