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        Introduction to Antibodies - Appendix

        互联网

        1629
         

        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)

        1. 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.
        2. Add 20 g granular paraformaldehyde and stir until it is dissolved.
        3. 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.
        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.

        1. Heat 250 mL double strength phosphate buffer stock solution (see above) to 140� (60�) in a beaker with a disposable stir bar.
        2. Add 20 g granular paraformaldehyde and 10 g gluteraldehyde and stir until it is dissolved.
        3. 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)

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

        Bouin� Fluid

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

        Carnoy� Fixative

        1. 10 mL of glacial acetic acid
        2. 30 mL of chloroform
        3. 60 mL of absolute alcohol (100% Ethanol)
        4. Mix

        PLP (Periodate-lysine-paraformaldehyde) Fixative

        1. Dissolve 7.3 g of lysine monohydrochloride in 200 mL of ddH2 O.
        2. Adjust pH to 7.4 with 0.1 M Na2 HPO4 (Na2 HPO4 ? H2 O 17.8 g/L) NOT phosphate buffer!
        3. 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.
        4. 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

        1. 100 mL acetone
        2. Add 100 mL methanol
        3. 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

        1. Pipet 450 � buffer into a tube.
        2. Add 50 � antibody solution, and mix. This gives a 1:10 dilution of the antibody.
        3. Label tubes A through M for 1:50, 1:100, 1:200, 1:400, etc. to 1:51,200 dilutions.
        4. 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).
        5. Pipette 400 � of 1:10 antibody dilution into tube A (which contains 1600 � buffer). Mix well. This results in a 1:50 antibody dilution.
        6. Take 1000 � of antibody sample from Tube A and add to Tube B (which contains 1000 � buffer). Mix well.
        7. 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 IgG2a ++++ ++++
        Mouse IgG2b +++ +++
        Mouse IgG3 ++ +++
        Mouse IgGM +/- -
        Pig Ig +++ +++
        Rabbit Ig ++++ +++
        Rat IgG1 - +
        Rat IgG2a - ++++
        Rat IgG2b - ++
        Rat IgG2c + ++
        Rat IgGM +/- -
        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


        <center><a name="APPENDIX_E"> <font><font><font><font><font><font><font><font><font><font><font></font></font></font></font></font></font></font></font></font></font></font></a><font><font><font><font><a name="APPENDIX_E"> </a></font></font></font></font><a name="APPENDIX_E"> <p> <font><font><font><font><font><font><font><font><font><font><font>[2] 下一页 </font></font></font></font></font></font></font></font></font></font></font></p> </a></center>
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