Introduction to Antibodies - Appendix

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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.

Tube	Sample to be diluted	Volume of Sample	Volume of Buffer	Resulting Dilution
A	1:10	400 �	1600 �	1:50
B	1:50	1000 �	1000 �	1:100
C	1:100	1000 �	1000 �	1:200
D	1:200	1000 �	1000 �	1:400
E	1:400	1000 �	1000 �	1:800
F	1:800	1000 �	1000 �	1:1,600
G	1:1,600	1000 �	1000 �	1:3,200
H	1:3,200	1000 �	1000 �	1:6,400
I	1:6,400	1000 �	1000 �	1:12,800
J	1:12,800	1000 �	1000 �	1:25,600
K	1:25,600	1000 �	1000 �	1:51,200
L	1:51,200	1000 �	1000 �	1:102,400
M	1:102,400	1000 �	1000 �	1:204,800

APPENDIX C Protein A/G Binding Affinities

Species	Immunoglobulin	Protein A	Protein G
Bovine	Ig	++	++++
Chicken	Ig	-	+
Goat	Ig	+/-	++
Guinea Pig	Ig	++++	++
Hampster	Ig	+	++
Mouse	IgG₁	+	++
Mouse	IgG_2a	++++	++++
Mouse	IgG_2b	+++	+++
Mouse	IgG₃	++	+++
Mouse	IgG_M	+/-	-
Pig	Ig	+++	+++
Rabbit	Ig	++++	+++
Rat	IgG₁	-	+
Rat	IgG_2a	-	++++
Rat	IgG_2b	-	++
Rat	IgG_2c	+	++
Rat	IgG_M	+/-	-
Sheep	Ig	+/-	++

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

1. ALKALINE PHOSPHATASE

Substrate	Buffer/ Second Substrate	Reagent to stop reaction	Soluble or Insoluble Product	Color of Product	Wavelength for quantitation
p-Nitrophenyl Phosphate (pNPP)	Na₂ CO₃ , pH 9.8 with MgCl₂	NaOH, 2M	Soluble	Yellow	405 nm
Bromochloroindolyl Phosphate-Nitro blue Tetrazolium (BCIP/NBT)	NaCl, MgCl₂ , Diethanolamine	EDTA Purple	Insoluble	Black	N/A

2. HORSERADISH PEROXIDASE

Substrate	Buffer/ Second Substrate	Reagent to stop reaction	Soluble or Insoluble Product	Color of Product	Wavelength for quantitation
3,3',5,5'-Tetramethyl- benzidine (TMB)	30% Hydrogen Peroxide (H₂ O₂ )	1 M Sulfuric Acid (H₂ SO₄ )	Soluble	Yellow	450 nm
o-Phenylene Diamine (OPD)	Citrate Phosphate Buffer, 0.02% H₂ O₂	Sulfuric Acid (H₂ SO₄ )	Soluble	Orange-Brown	492 nm
2,2'-azinodiethyl- benzthiazoline sulfonate(ABTS)	Citrate Phosphate Buffer, 30% H₂ O₂	20% SDS / 50% DMF	Soluble	Green	410 nm, 650 nm
Chlornaphthol	30% H₂ O₂	PBS	Insoluble	Blue-black	N/A
3-Amino-9-ethylcarbazole (AEC)	30% H₂ O₂	PBS	Insoluble	Red	N/A
Diaminobenzidine (DAB)	30% H₂ O₂	PBS	Insoluble	Brown	N/A

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