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It is a kind of immunochemical techniques which is used to detect a protein immobilized on a matrix. The target protein can be in a crude extract or a more purified preparation and the monoclonal or polyclonal antibody against this protein is necessary to help us to recognize the antigen. As in the Figure, soluble antigens (the target protein) may be separated by electrophoresis based on its molecular weight (SDS/PAGE), size and charge (nondenaturating gel electrophoresis or isoelectric point (isoelectric focusing). After the separation, the proteins are transferred from the gel to a PVDF membrane. Once on the membrane antibodies (first antibodies) can be used to probe for the presence of particular protein because of the specifically binding of antigen with against it. Non-specific binding site can be  blocked using other non-specific protein such as bovine serum albumin before adding first antibody to avoid non-specific binding. Protein transfer is most commonly accomplished by electrophoresis This procedure is called electrophoretic blotting. The two common electrophoretic methods are: $ Semi-dry blotting, in which the gel and immobilizing matrix are sandwiched between buffer-wetted filter papers through which a current is applied for 10-30 minutes. $ Wet (tank) blotting, in which the gel-matrix sandwich is submerged in transfer buffer for electrophoresis, which may take as little as 45 minutes or may be allowed to continue overnight We only describe wet blotting here, since it permits greater flexibility without being significantly more expensive in time or materials. The detection of target protein is using a second antibody, which can recognize the first antibody. Typically, the second antibody is purchased already conjugated to a labeling agent such as the enzyme horseradish peroxidase. This marker is then visualized by a colorimetric reaction catalyzed by the enzyme which yields a colored product that remains fixed to the membrane. Thus, it is possible to recognize first antibody through recognizing second antibody, and then identify the position of target protein. Other detection systems include alkaline phosphatase and 125I labels. 0Materials0 $ Apparatus: Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membraneMillipore Immobion-P #IPVH 000 10 , Whatman 3MM paper, Additional Tools: Forceps, sponge pad, scissor, gloves, small plastic or glass container, Shallow tray. $ Reagents: t$ 10x transfer buffer (1 L): 30.3 g Trizma base (0.25 M), 144 g Glycine (1.92 M), pH should be 8.3; without adjustment. u$ 1x transfer buffer (2 L): 400 ml Methanol, 200 ml 10x transfer buffer, 1400 ml water. v$ TBS buffer: Add 1.22g Tris (10 mM) and 8.78g NaCl(150 mM) to 1L distilled water and adjust pH to 7.5 with HCl. w$ TTBS buffer: 1L TBS buffer add 0.5<>@F H J LR^`bî}jS};.jh}h%B*OJQJU^JaJph333,h}hw6_B*CJH*OJQJ^JaJph333$h}hw6_0JCJOJQJ^JaJ,h}hw6_B*CJOJQJ^JaJo(ph3332jh}hw6_B*CJOJQJU^JaJph333)h}hw6_B*CJOJQJ^JaJph333)h}h B*CJOJQJ^JaJph333"h B*OJQJ^JaJo(ph333)h 5B* CJOJQJ\^JaJph\>`f:QQQQdhgdw6_gduYdhgduY ~WD`~gduY$a$gd}Qbdfvxbdpr 68DFfhlnbȷȷȷȝȷȷȷȷȷȷȷȷȷȉ{kYk"huYB*OJQJ^JaJo(ph333huYB*OJQJ^JaJph333huY0JOJQJ^JaJ&huYB*CJOJQJ^JaJo(ph3332jyh%h%B*CJOJQJU^JaJph333 h%hw6_B*CJaJo(ph333)h%hw6_B*CJOJQJ^JaJph333"hw6_B*OJQJ^JaJo(ph333hw6_B*OJQJ^JaJph333"*%,%' '6'8'&((((())*4,4.44455@6B6f7h7~8899:::::====@@FEHEJELEFFBFIJx2jShhuYB*CJOJQJU^JaJph333huYB*CJaJo(ph333#huYB*CJOJQJ^JaJph333"huYB*OJQJ^JaJo(ph333.j]hhuYB*OJQJU^JaJph333UhuYB*OJQJ^JaJph333"huYB*OJQJ^JaJo(ph333/ml Tween 20 (0.05%). x$ First antibody: antibody against the target protein. y$ Second antibody: goat anti-rabbit-HRPhorseradish peroxidase . z$ 3% Blocking buffer (0.5 L): Add 15mg Bovine serum albumin in TBS buffer to final volume 0.5 L, keep at 4C to prevent bacterial contamination. {$ 0.5% Blocking buffer (0.5 L): Add 2.5mg Bovine serum albumin in TBS buffer to final volume0.5 L, keep at 4C to prevent bacterial contamination. |$ Developing reagent: 1ml chlonoaphthol solution (30mg/ml in methanol), add 10 ml methanol, add TBS buffer to 50 ml and add 30 ul 30% H2O2. }$ Staining buffer: Add 1g amido black 18B (0.1%), 250ml isopropanol (25%) and 100 ml acetic acid (10%) to distilled water with final volume 1L. ~$ Destaining buffer: Add 350ml isopropanol (35%) and 2 ml acetic acid(2%) to distilled water with final volume 1L.  0Procedure0 $. Separation of Protein Run an electrophoretic separation of known antigenic proteins. The method of separation decided by the characters of target protein, but for sufficiently transferring, the most common method is SDS-PAGE. After separation, remove upper side of sample wells with a razor blade. Notching bottom right-hand corner of gel for orientation and put gel in transfer buffer until ready to use. $. Electrotransfer t$ Preparation of membrane Cut a piece of PVDF membrane (Millipore Immobion-P #IPVH 000 10) according to the size of gel. Incubate in methanol for about 1 min on a rocker at room temp. Remove methanol and equilibrate membrane in 1x transfer buffer until ready to use. u$ Arrange gel-membrane sandwich In a shallow tray, open the transfer cassette. Put a well-soaked sponge pad on the black piece of the transfer cassette and a wetted 3MM paper on the sponge pad. Place the gel on the paper and arrange well so that all air bubbles are removed. Lay the PVDF membrane on the top of gel and remove any air bubbles. Place a wetted sheet of 3MM paper over the PVDF membrane and remove the bubble. Covered with the second well-soaked pad. Close the sandwich with the white piece of the cassette. Mount the sandwich in the transfer tank; put the black sides near the black side of the device. Fill the buffer tank with the transfer buffer.  v$ Electrotransfer: Attach the electrodes. Set the power supply to 100V (constant voltage) for 1h at 4C. $. Immunodetection t$ Membrane staining Disconnect transfer apparatus, remove transfer cassette, and peel 3MM paper from membrane. Remove the membrane to a small container. Add 10 ml TBS buffer and wash for short time. Cut out one stripe with 5mm width and put in another clean container. Stain this stripe in staining buffer for 1 min. Destain for 30 min in destaining buffer to check whether protein has been transferred from gel to membrane or not. u$ Membrane blocking and washing For other part of membrane, pour off TBS buffer. Add 3% blocking buffer rock gently for at least 1 h. Pour off 3% blocking buffer and rinse briefly with TBS buffer three times, 5 minutes for per time. v$ First antibody Pour off TBS buffer. Add first antibody at appropriate dilution in 10 ml 0.5% blocking buffer. Rock gently for at least 1 h; pour off first antibody solution from membrane and wash twice for 10 minutes with TTBS buffer. w$ Second antibody Pour off TTBS buffer. Add second antibody at appropriate dilution in 5 ml 0.5% blocking buffer. Rock gently for 30 min, pour off second antibody solution from membrane and wash twice for 10 minutes with TTBS buffer. x$ Detection Pour off TTBS buffer from membrane and add developing reagent, Rock PVDF gently, monitoring development. When the bands can be seen clearly, stop development by washing membrane with distilled water for 30 minutes with 3 changes. 0Result0 Check the bands on membrane, the band with blue-purple color corresponding to the target protein. BFDFIItKvKPMRM$O&OQQQQƺh%hw6_CJaJo()h%hw6_B*CJOJQJ^JaJph333 huYo(#huYB*CJOJQJ^JaJph333huYB*CJaJo(ph333 0182P. A!"#$%S yDd@  C A1Ry% -hY178OxD|Fx% -hY178OJFIF``ExifII*C    $.' 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