Editing PKA ELISA

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

<br>ELISA:<br>1. Discard the blocking solution and wash the plate <strike>two</strike> 3 times with <strike>TBST</strike> PBST, 200 ul/time;<br>2. Add 100 ul serum (1:2,000 diluted with sample diluent solution) to each well; for control add 100 ul sample diluent solution;<br>3. <strike>37°C</strike>, RT for 1 hour;<br>4. Discard serum;<br>5 .Wash wells three times with wash buffer, 200ul/time;<br>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;<br>7. RT, 1 hour;<br>8. Discard the 2nd antibody;<br>9. Wash wells with wash buffer 6 times, 200 ul/time, 5 min incubation;<br>10. Add 100 ul SureBlue Reserve ELISA Substrate from KPL, catalog number: SPL-SBR;<br>11. RT, 30 min;<br>12. Add 100 ul TMB Stop Solution from KPL, catalog number: 50-85-05;<br>13. Record OD<sub>450</sub> or record OD<sub>450</sub> and use OD<sub>650</sub> as reference. 

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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. 

<strike><u>TBS</u></strike> '''PBS solution''': <strike>Tris</strike> Phosphate Buffered Saline,<strike>50 mM Tris, 150 mM NaCl, pH 7.4 </strike> 

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

<br>'''Wash buffer''':<strike>TBST (TBS&nbsp;</strike> PBS containing 0.05% Tween 20)<br> 

'''10X TBS:'''<br>* 200 ml of ultrapure water<br> * 500 ml of 1 M Tris.Cl pH 7.4<br> * 300 ml of 5 M NaCl (or use 90 grams of NaCl crystals)<br><br>'''TBST:''' Dilute to 1X TBS, add '''0.05%''' Tween-20 (250uL Tween-20 in 500mL 1XTBS ---&gt; 1X TBST)<br> 

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

'''Stop Solution:''' 2M Sulfuric Acid <br> 

[[Category:Protocols]]