Introduction to Antibodies - Appendix

Caution: Formaldehyde is toxic and should be handled with caution under a chemical fume hood. Consult Material Safety Data Sheets for proper handling of all laboratory chemicals.

4% Paraformaldehyde (PFA)

Heat 250 mL of double strength phosphate buffer stock solution (see step 4) to 140� (60�) in a beaker with a disposable stir bar in a hood. Add 20 g granular paraformaldehyde and stir until it is dissolved. Add 250 mL deionized water and filter the solution into a container placed on ice. The solution is ready when cold. Adjust pH to 7.0?.4. Double Strength Phosphate Buffer Stock solution is prepared by dissolving 7.7 g NaOH and 33.6 g NaH2 PO4 in 1 liter deionized water.

4% Paraformaldehyde with 2% Gluteraldehyde.

Heat 250 mL double strength phosphate buffer stock solution (see above) to 140� (60�) in a beaker with a disposable stir bar. Add 20 g granular paraformaldehyde and 10 g gluteraldehyde and stir until it is dissolved. Add 250 mL deionized water and filter the solution into a container placed on ice. The solution is ready when cold. Adjust pH to 7.0?.4.

Buffered Formaldehyde (Formalin)

Dissolve 32.5 g Na2 HPO4 and 20 g NaH2 PO4 in 4.5 L deionized water. Add 500 mL 40% Formaldehyde. Mix; Adjust pH to 7.0?.4.

Bouin� Fluid

Picric Acid (standard aqueous solution) 75 mL. Formalin (40% aqueous Formaldehyde) 20 mL Glacial Acetic Acid 5 mL. Mix

Carnoy� Fixative

10 mL of glacial acetic acid 30 mL of chloroform 60 mL of absolute alcohol (100% Ethanol) Mix

PLP (Periodate-lysine-paraformaldehyde) Fixative

Dissolve 7.3 g of lysine monohydrochloride in 200 mL of ddH2 O. Adjust pH to 7.4 with 0.1 M Na2 HPO4 (Na2 HPO4 ? H2 O 17.8 g/L) NOT phosphate buffer! Complete volume to 400 mL with 0.1 M phosphate BUFFER (!!) pH 7.4. This lysine-phosphate buffer keeps for 2? days in the refrigerator, but can be frozen in aliquots for longer storage. Just before use, mix 375 mL lysine-phosphate buffer with 100 mL 20% Formaldehyde and top to 500 mL with ddH2 O. Add 1.06 g Sodium periodate (NaIO4 ) and mix well. The PLP fixative must be used within a maximum of 2 hrs.

Final concentrations: Lysine 75 mM, Formaldehyde 4%, Sodium periodate 10 mM. (Note: Some PLP formulations in literature also use 2% paraformaldehyde )

Acetone/Methanol Fixative

100 mL acetone Add 100 mL methanol Mix well. Use fresh. 50?0 solution is used at room temperature or -20�

APPENDIX B Making Serial Antibody Dilutions

Reagents/Equipment:

• PBS or other appropriate buffer.
• Small capped tubes
• Pipets capable of accurate delivery of 200 mL and 1000 mL volumes

Keep buffer and tubes in ice

Pipet 450 � buffer into a tube. Add 50 � antibody solution, and mix. This gives a 1:10 dilution of the antibody. Label tubes A through M for 1:50, 1:100, 1:200, 1:400, etc. to 1:51,200 dilutions. Pipet 1600 � of dilution buffer into tube A (to become a 1:50 dilution). Pipette 1000 � (1.0 mL) of dilution buffer into tubes B through M (to become 1:100 - 1:102,400 dilutions). Pipette 400 � of 1:10 antibody dilution into tube A (which contains 1600 � buffer). Mix well. This results in a 1:50 antibody dilution. Take 1000 � of antibody sample from Tube A and add to Tube B (which contains 1000 � buffer). Mix well. Take 1000 � of antibody sample from Tube B and add to Tube C (which contains 1000 � buffer), etc. Mix well.

APPENDIX C Protein A/G Binding Affinities

APPENDIX D Enzyme Substrates for ELISA Testing (soluble substrates) and Blotting (insoluble substrates)

1. ALKALINE PHOSPHATASE