Western Blot (Immune Imprinting) experimental method steps.

Western Blot (Immune Blot)
consists of the following 4 basic steps:
n sample preparation
n electrophoresis separation
n protein membrane transfer
n immunobreeding and visible Chroma protein
solution and
reagent
n1X phosphate buffer (PBS)
nModified RIPA buffer
Tris-HCl: 50 mM, pH 7.4; NP-40: 1%; Na-deoxycholate: 0.25%; NaCl: 150 mM; EDTA: 1 mM; PMSF: 1 mM; Aprotinin, leupeptin, pepstatin: 1 microgram/ml each; Na3VO4: 1 mM; NaF: 1 mM
n1X SDS sample buffer
62.5 mM Tris-HCl (pH 6.8 at 25 degrees C), 2% w/v SDS, 10% glycerin, 50 mM DTT, 0.01% w/v Bromphenol blue
n transfer buffer
25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.3)
n10X Tris buffer salt (TBS)
Prepare 1L 10X TBS: 24.2 g Tris base, 80 g NaCl; 7.6
n skimmed milk powder or BSA
n methanol
nTBS/T buffer
1X TBS, 0.1% T ween-20
n closed buffer (TBS/T)
1X TBS, 0.1% Tween-20 plus 5% w/v skimmed milk powder or BSA
n resistance. Diluted
1X TBS, 0.1% Tween-20 plus 5% BSA (multi-resistance) or 5% skimmed milk powder (monoantigen)
Note: In general, BSA is recommended for multiclonal
antibody
, skimmed milk powder for monoclonal antibodies, which results in a high signal-to-noise ratio. The dilution of the antibody is determined by reference to the antibody instructions or by the experiment.
n pre-contaminated
protein
Marker, can be used to monitor the efficiency of the transfilmsample preparation the original sample can be cells, tissues,
culture
on the clear, immuno-precipitation or affinity purified protein, the following is the qualitative testing purpose of protein cell samples treatment methods, the rest of the sample preparation methods refer to the relevant literature.
1. Culture cells or drug treatment.
2. Discard
the
, rinse the cells 2 times with 1X PBS to remove the remaining culture.
3. Add 1X SDS sample buffer (6-well plate, 100 sl /w or 75 cm2 plate, 500-1000 sl/bottle), scrape off the cells and transfer to the Ep tube. Note: Operate on ice.
4. Ultrasound 10 to 15 seconds
DNA
reduce sample stickiness.
5. Bring the sample to a boil for 5 minutes.
6. Centrifuged 12000g, 5 min, cleared.
7. Electrophoresis separation: sample 15 to 20 μl to SDS-PAGE glue (10 cm x 10 cm) electrophoresis.
to quantify the expression level of a protein, use RIPA lysate cells (1 ml per 107 cells/100 mm dish/150 cm2 flask) to collect lysate into the centrifuge tube and mix 4 to 4 to 4 15min, 14000g centrifugation 15min (4 oC), discard precipitation, use Bradford method or other protein assay method to determine the concentration of the upper and middle proteins to adjust the volume of the upper sample and the upper sample amount, Western hybridization also need to set internal or external reference, usually with beta-actin.
Note: General sample 20 to 30 μg is sufficient, such as the protein to be tested as a low abundance protein, can increase the sample amount to 100 sg, but electrophoresis strip easy to drag tail, can prepare subcellular parts or use more sensitive detection methods.electrophoresis separation (referring to the SDS-PAGE electrophoresis method)transfer membranehybrid membrane selection is an important link in determining the success or failure of Western blot. The appropriate material, aperture and specification of the hybrid film should be selected according to the hybridization scheme, the characteristics of the transferred protein and the molecular size. There are two main types of membranes for Western blot: the cellulose nitrate membrane (NC) and the PVDF membrane. NC membrane is the standard solid-phase support of protein imprinting experiment, in the low ion transfer buffer environment, most negatively charged proteins will be hydrophobic and high affinity binding with the membrane, but under the action of non-ion type decontamination agent, the binding protein can also be extinct. Depending on the molecular weight of the transferred protein, NC membranes with different apertures are selected. Because as the membrane aperture decreases, the stronger the membrane's binding to the low molecular weight protein. NC membranes of both sizes, 0.45 m and 0.2 m, are commonly used. Proteins larger than 20kD can be used in a membrane of 0.45 m, proteins smaller than 20kD can use a membrane of 0.2 m, such as a membrane of 0.45 m will occur "Blowthrough" phenomenon. PVDF membrane sensitivity, resolution and protein affinity are higher than conventional membranes and are ideal for low molecular weight protein detection. However, the PVDF membrane must be soaked with pure methanol for 1-5 seconds before use.
commonly used transfer methods for proteins are two main: slot wet turn and semi-dry transfer. The former is easy to operate and the transfer efficiency is high, while the latter is suitable for protein transfer of large glue, using less buffer. The following are the procedure for slotting wet turns.
1. Dip the glue in the transfer buffer to balance 10min.
note: if the detection of small molecular proteins, you can omit this step, because small molecular proteins easily spread out of the glue.
2. Cut the film and filter paper 6 pieces according to the size of the glue, put in the transfer buffer to balance 10min. If using PVDF film to soak with pure methanol saturation for 3-5 seconds.
3. Assembly transfer sandwich: sponge ®3 layers of filter paper ®glue® membrane®3 layers of filter paper® sponge, each layer put away, with a test tube to get out of the bubble. Remember: the glue is on the negative side (black side).
4. Place the transfer slot in an ice bath, place the sandwich (black facing the black side), add the transfer buffer, plug in the electrode, 100V, 1h (current is about 0.3A). Note: Check again that the sandwiches and electrodes are properly assembled and that the power supply is switched on.
the end of the transfilm, cut off the power supply and remove the . Immune hybridization and color 1. Wash 5min with 25 ml TBS, room temperature, shake.
2. The film is placed at 1h in a 25 ml closed buffer, at room temperature, shaking.
3. 15ml TBS/T wash 3 times (5 min/T).
4. Add a suitable dilution resistance, incubate at room temperature 1-2h or 4 degrees C overnight, shake slowly.
5. 15 ml TBS/T wash 3 times (5 min/T).
6. Add a suitable dilution of alkaline
phosphatase
(AP) or spicy root peroxidase (HRP) labeled secondary resistance, incubate at room temperature 1h, slowly shake.
7. 15 ml TBS/T wash 3 times (5 min/T).
8. 15 ml TBS wash 1 time.
9. Protein detection (color or luminescence, according to the appropriate reagent instructions).: 1. Wear gloves in operation and do not touch the membrane with your hands.
2. The PVDF film should not be soaked in methanol for more than 5 seconds.
3. For example, the membrane of 0.2 m applied to proteins less than 20kD can be detected, and the balancing step at the time of transfer can be omitted.
4. Some
antigens
antibodies can be washed away by Tween-20, at which point 1.0% BSA can be used instead of Tween-20.
5. Choice of closed agents: 5% skimmed milk/TBS or PBS: ability to interact with certain antigens to mask antibody binding capacity;
6. If you use 0. 1% Tween 20, 0.02% NaN3 in PBS or TBS as a closure agent and antibody diluent, antibody detection can be carried out protein staining.
to detect both large molecular weight and small molecular protein, it is best to separate the protein with gradient glue. .