LaCava Research Wiki

AIM/HYPOTHESIS

Detection of ORF2p in endogenous sample has not been done successfully. Previous results, where we can detect ORF1p after a-ORF2p IPs (01/07/20 - Capturing ORF2 with rabbit anti-ORF2 beads from N2102Ep and MT302 and 01/22/20 - Capturing ORF2p with anti-ORF2 beads from N2102Ep) give us confidence that ORF2p is present in endogenous cell line N2102Ep. To detect it, we will pursue an approach where we blot with biotinylated secondary abs and subsequently with streptavidin-polyHRP.

See https://onlinelibrary.wiley.com/doi/full/10.1002/elps.201900059

If we are indeed able to detect ORF2p in an endogenous context, we will establish the endogenous ORF2p interactiome by shotgun m/s (samples in quadruplicates, IgG controls), compare to endogenous ORF1p interactome and additionally try to detect ORF2p with the PLA assay.

MATERIALS

METHODS

Dilutions might need to be adjusted. For now I will go with;

Streptavidin – Dissolved in 100 µL H2O to 10 mg/mL stock as manual instructed. Diluted 1:2000. In Mishra et al., 2019 they use 50 µg/mL (1:200), however, the spec sheet of the Streptavidin states that 1 mg of Streptavidin can bind 19 g (!) of biotin. Hence I decided to go for 1:2000 as a start. Stored with antibodies in 2nd refrigerator, box #1.

Biotin – We got 1 g of powder. I could not find info on its stability in solution, so for now we can just make it fresh each time. In Mishra et al., 2019 they use 0.25 mg/mL in TBST. I suppose this could also be lowered, but since we have plenty I will just use this concentration.

1° - 1:500 (as in previous blots by Hua)

2° - 1:1000, I used the KPL antibody as specified in the materials

Strep-PolyHRP80 – 1:10.000 (might need diluting even more, as in the previously mentioned paper they state that 1:40.000 is often sufficient)

Blotting with Biotinylated secondary ab and PolyHRP conjugated streptavidin

Steps:

1. Strip blot of a-ORF2p IPs

2. Block using 5% not-fat milk in 1x TBST for 1hr at RT

3. Wash 1x 5’ with 1x TBST

4. Check if stripping of the blot was successful using chemiluminescence

5. Wash 1x 5’ with 1x TBST

6. Block endogenous biotin with Streptavidin (5 µg/mL in 1x TBST + 5% BSA) for 1h at RT

7. Wash 3x 5’ with 1x TBST

8. Block exogenous Streptavidin with exogenous biotin (0.25 mg/mL in 1x TBST + 5% BSA) for 1h at RT

9. Wash 3x 5’ with 1x TBST

10. Incubate with primary antibody dissolved in 1x TBST + 5% BSA at 4°C o/n

11. Wash 3x 5’ with 1x TBST

12. Incubate with biotinylated secondary antibody dissolved in 1x TBST + 5% BSA at for 1hr at RT

13. Wash 3x 5’ with 1x TBST

14. Incubate with Strep-PolyHRP80 dissolved in 1x TBST + 5% BSA

15. Wash 3x 5’ with 1x TBST

16. Detect by chemiluminescence

RESULTS

Reblotting of 01/07/20 - Capturing ORF2 with rabbit anti-ORF2 beads from N2102Ep and MT302

1) Marker
2) Input_N2102Ep_25ug
3) FT_N2102Ep_25ug
4) Input_MT302_25ug
5) FT_MT302_25ug
6) Input_MT302_2.5ug
7) FT_MT302_2.5ug
8) Marker (not shown)
9) Space
10) E_N2102Ep_19ul (95%, 950mg)
11) Space
12) E_MT302_1ul (10%, 5mg)
13) Marker (not shown)

Original

Anti-ORF2 Western (Clone 5-5), high sensitivity, 30s exposure, auto tone

Anti-ORF2 Western (Clone 5-5), super sensitivity, 5m exposure, auto tone

New

Left panel (60 sec ultra, no edit)

Right panel (120 sec high, no edit)

Reblotting of 01/22/20 - Capturing ORF2p with anti-ORF2 beads from N2102Ep

1) Marker
2) Input_2102Ep_Gerald_NP40_25ug
3) FT_2102Ep_ Gerald_NP40_25ug
4) Input_N2102Ep_RU_NP40_25ug
5) FT_N2102Ep_RU_NP40_25ug
6) Input_N2102Ep_RU_NP40_25ug
7) FT_N2102Ep_RU_NP40_25ug

Skip Input and FT of HeLa in order to fit all samples in 15-well gel

8) Input_MT302_2.5ug
9) FT_MT302_2.5ug
10) Marker
11) E_2102Ep-Gerald_NP40
12) E_N2102Ep_RU_NP40
13) E_N2102Ep_RU_L1
14) E_HeLa-Gerald_NP40
15) E_MT302_L1 (10% of Elution)

Original

1 min, super, auto tone

New

100 sec, super

DISCUSSION

I think the main conclusion for now is that at least the method works. MT302 shows obvious bands at ORF2. The failure to detect ORF2 in N2102Ep might, as discussed during the group meeting, have to do with the stripping of the blot.

I propose the following;

- Do a fresh a-ORF2 IP with 500mg, 250 mg, 100 mg of N2102Ep (Hua did 1g), with 10ul of beads per 50mg. Take along 50mg of MT302 as positive control. Load a gel as follows;

1) ladder
2) N2102Ep 100mg input
3) N2102Ep 250mg input
4) N2102Ep 500mg input
5) N2102Ep 100mg FT
6) N2102Ep 250mg FT
7) N2102Ep 500mg FT
5) N2102Ep 100mg E
6) N2102Ep 250mg E
7) N2102Ep 500mg E
8) ladder
9) MT302 input
10) MT302 FT
11) MT302 E
12) ladder 

And follow the above protocol for detection.

- Check the signal amplification with the new method. This can be done solely with MT302 by loading serial dilutions on the gel, first blotting with the regular method (using clone 5.5 of a-ORF2) and then with the new method after stripping (to prevent of over-estimation of the amplification).