PKA ELISA

PKA-C alpha ELISA protocol
96 well plate coating:
1.In each well, add PKA-C alpha in TBS PBS solution 100 ul (2ug/ml);
2. 4°C overnight;
3. Discard the coating solution and wash the plate with 200 ul TBS PBS two times;
4. Add 200 ul blocking solution and store at 4°C overnight; the plate is ready for use.

ELISA:
1. Discard the blocking solution and wash the plate two 3 times with TBST PBST, 200 ul/time;
2. Add 100 ul serum (1:2,000 diluted with sample diluent solution) to each well; for control add 100 ul sample diluent solution;
3. 37°C, RT for 1 hour;
4. Discard serum;
5 .Wash wells three times with wash buffer, 200ul/time;
6. Add 100 ul 2nd antibody (1:10,000 in sample diluent solution); 2nd antibody for human serum is Peroxidase labeled Gt anti-human lgG (H+L) from KPL, catalog number is HRPGTXHU.05;
7. RT, 1 hour;
8. Discard the 2nd antibody;
9. Wash wells with wash buffer 6 times, 200 ul/time, 5 min incubation;
10. Add 100 ul SureBlue Reserve ELISA Substrate from KPL, catalog number: SPL-SBR;
11. RT, 30 min;
12. Add 100 ul TMB Stop Solution from KPL, catalog number: 50-85-05;
13. Record OD₄₅₀ or record OD₄₅₀ and use OD₆₅₀ as reference.

Note for wash: for each wash, leave the wash buffer in each well for at least 30 second, shake the plate gently several times during the washing period. Invert the plate over a sink and shake it vigorously. Drain residul buffer by tapping plate on filter paper.

TBS PBS solution: Tris Phosphate Buffered Saline,50 mM Tris, 150 mM NaCl, pH 7.4

Blocking or sample diluent solution: 1% BSA (albumin from bovine serum, Sigma catalog number A9418-50G), 10% glycerol in TBS PBS

Wash buffer:TBST (TBS  PBS containing 0.05% Tween 20)

10X TBS:
* 200 ml of ultrapure water
* 500 ml of 1 M Tris.Cl pH 7.4
* 300 ml of 5 M NaCl (or use 90 grams of NaCl crystals)

TBST: Dilute to 1X TBS, add 0.05% Tween-20 (250uL Tween-20 in 500mL 1XTBS ---> 1X TBST)

Carbonate Coating Buffer:
3.03 g Na2CO3,
6.0 g NaHCO3
1000 ml distilled water
pH 9.6,

Stop Solution: 2M Sulfuric Acid