Adjust trarsmembrane condition according to the molecular weight of target proteins.Discharge bubbles and put them into wet/electrical transfer tank.Put black plate(negative electrode) - fiber pad - filter paper - gel - PVDF membrane or NC membrane - filter paper - fiber pad - white plate(positive electrode) in order.Also immerse filter paper and fiber pad in the transfer buffer.Immerse and activate the membrane in the methanol for 5min.Select PVDF or NC membrane with different pore sizes according to the molecular weight of target proteins.Change 90v constant pressure electrophoresis to 120v till completing the process and the end of electrophoresis, after bromophenol blue indicator linearly moves to the junction between SDS-PAGE and separating gel.Ĥ.3.Add 1x electrophoresis buffer for enabling the electrophoresis.Also reserve a lane to add 5μL pre-stained protein marker for validating target molecular weight and level of trarsmembrane.Add the sample to be tested in each lane.Prepare separating gel with different concentration according to the molecular weight of target proteins.Try to keep the loading capacity of each sample is close to 10μL. Notes: Suggested loading amount of total protein for samples ready for detection is 50-100μg. Collect the supernatant to perform western blot or store at -20℃/-80℃.Perform the centrifugation at 12,000r/min for 2min.According to the percentage of 4:1, add 5×SDS loading buffer to boil for 10 minutes.Measure protein concentration by BCA method. 2S ultrasonic delay is set to ensure complete cell lysis and decrease the sample viscosity perform the centrifugation at 12,000r/min for 5min at 4℃ and then get the supernatant for testing protein concentration.Ĭell Sample Processing: Collect the cell sample Wash components like culture medium etc by precooling PBS Rest steps are the same as tissue sample processing. Tissue Sample Processing: Get the tissue sample ready for detection Wash blood on the tissue surface and internal impurities by precooling PBS Weigh and cut tissue sample Add appropriate RIPA lysate complex(add 10μL PMSF into 1mL RIPA lysate ) to perform the homogenate lysis Then, perform the shock lysis on the ice for 60min Under 35%-40% power, the sample is processed through ultrasonic treatment for 1min(in the ice bath condition). Operation steps for western blot: protein extraction, content measurement, SDS-PAGE electrophoresis, immune response, chemiluminescence, gel image analysis. Commonly used types are DAB substrate staining and ECL substrate which is usually applied in academic publications. Western blot includes the following types according to the chromogenic method: autoradiography, ECL substrate, ECF substrate, DAB substrate staining. Western blot is also applied in testing protein expression level.įigure 1. Finally, specific target gene expressed protein components are tested by staining of substrate or autoradiography.
The protein or polypeptide used as an antigen on the solid phase carrier triggers an immune response for the relevant antibody, and then reacts with secondary antibodies labeled by enzymes or isotopes. The solid phase carrier adsorbs proteins in the noncovalent form and can keep polypeptide type separated by electrophoresis and biological activity unchanged. Protein samples separated by PAGE are transferred to solid phase carrier (e.g. The labeled secondary antibody is used for staining.
Similar to southern blot or northern blot hybridization technique, but western blot uses polyacrylamide gel electrophoresis (PAGE).
Currently, western blot is widely used in expression of gene at protein level, antibody activity test and early diagnosis of diseases etc. Western blot is a kind of protein detection technique, which transfers cell or total protein separated by electrophoresis from gel to solid phase carrier like NC membrane or PVDF membrane and then test a specific antigen by specific antibody.
This protein method was first known as western blot in the Analytical Biochemistry written by Neal Burnette in 1981. Western blot was proposed by Harry Towbin from Switzerland Friedrich Miescher Institute in 1979. Then, the information of specific protein expressed in analyzed cell or tissue is obtained. Cell or biological tissue samples processed by gel electropherosis are stained by specific antibodies. Western blot is a common experimental method in molecular biology, biochemistry and immunogenetics. Keywords: Western Blot Step by Step, Protein Analysis Methods, Molecular Biology Techniques, Western Blot Antibody, FineTest Antibody 1. Especially, there are great influences and development space in medical applications. Abstract: Western blot has been widely applied in modern biological research for qualitative and semi-quantitative protein analysis since the invention.
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