Template:SBB-Protocols ELISA

Materials

Buffers
1. TBST

For 500mL of 10X TBS:
* 12.114 g Tris (FW 121.14)
* 43.83 g NaCl (FW 58.44)
Autoclave
Dilute to 1X and add 0.05% Tween-20 (250uL tween-20 in 500mL TBST)

2. Blocking Buffer = 2% BSA in TBST

 10% BSA = 10g/100mL

3. 1-Step Ultra TMB ELISA
4. 2M Sulfuric Acid

Plate
Nunc Lockwell Maxisorp (strips of 8 pop out)

Neg. Controls

1. non-mediator expressing cells with all antibodies (pMLLCA-??) pir+ 116
2. no detecting primary with mediator proteins (mixture of all?)

Preparing Cell Lysate

The 1-2 days before the assay: Pick colonies into 1mL 2YT + antibiotics and let grow to saturation.
Transfer 2uL of saturated cell culture into 1mL 2YT + arabinose + antibiotics and grow to saturation again.
Pellet. Spin 10,000g for 10 min.
Add 100uL room temperature bug buster. Vortex or pipette to resuspend and move into 96 well PCR plate.
Let incubate at room temperature (while shaking) for 10-20 min.
Pellet debris
Use 70uL lysate + 30uL blocking buffer in ELISA. Use 10uL and 1uL in BCA assay.

BCA Assay

1. Prepare BCA working reagent

Pierce BCA Kit: Reagent A and Reagent B (50 vol Reagent A + 1 vol Reagent B, use 200 uL per well).

2. Load 2uL sample+8uL ddH20 and 10uL standards into 96-well plate in duplicate 3. Add 200 ul BCA working reagent mix to each well 4. Incubate at 37C for 30 min 5. cool to room temp 6. Measure absorbance at 562nm

Procedure

1. Dilute primary capture antibody into TBST
2. Incubate 100uL for 1 hour at room temperature or overnight at 4C
3. Wash (3X) for 10 min with 200uL Blocking Buffer
4. Block 1 hour
5. Prepare cell lysate (see above) Run BCA Assay
6. Incubate Cell lysate for 1 hour
7. Wash (3X) for 10 min with 200uL Blocking Buffer
8. Dilute primary detecting antibody into Blocking buffer
9. Incubate 100uL detecting antibody for 1 hour at room temperature or overnight
10. Wash (3X) for 10 min with 200uL Blocking Buffer
11. Dilute secondary (HRP-conjugated) antibody into Blocking buffer
12. Incubate 100uL for 1 hour at room temperature or overnight
13. Wash (3X) for 10 min with 200uL Blocking Buffer
14. Bring TMB to room temperature
15. Add 50-100uL of TMB to each well
16. Incubate for 15-30 min at room temperature or until color change
17. Add 50-100uL of 2M Sulfuric Acid
18. Measure absorbance at 450nm