0:00 when working with proteins one cannot
0:03 get around the technique
0:05 called protein immunoblot or
0:08 simply the western blot western blotting
0:11 is a method to confirm the presence of a
0:14 specific target protein
0:16 in a sample before the analytical
0:19 technique western blood comes into play
0:22 everything starts with an sds page
0:25 upon treatment with sds all proteins in
0:29 a sample
0:29 are denatured and covered by negative
0:32 charges
0:33 the proteins have a similar master
0:35 charge ratio
0:37 and travel through the gel to the
0:39 positively charged anode
0:42 this allows to separate proteins
0:44 according to their molecular weight
0:47 after staining the polyacrylamide gel
0:50 protein bands become visible marker
0:53 proteins
0:53 as a reference help to determine the
0:56 molecular weight
0:57 of proteins in the sample this band here
1:01 for example
1:02 contains proteins with a molecular
1:04 weight of approximately
1:06 60 kilodalton however that will not
1:09 reveal
1:10 which proteins are present in this
1:12 sample it could be the protein of
1:14 interest which has a molecular weight in
1:16 this range
1:17 but the band could have also been caused
1:20 by
1:20 other proteins to confirm the presence
1:23 of a specific protein
1:25 a western blood has to be performed the
1:27 detection of the protein of interest is
1:30 based on
1:30 antibodies designed to exclusively bind
1:34 epitopes of
1:35 the target protein the western blot
1:37 setup starts with the gel containing
1:40 the separated proteins this is placed on
1:43 top of a membrane
1:45 often in nitrocellulose membrane
1:48 this membrane is capable to bind large
1:51 amounts of protein
1:53 later the idea is to transfer the
1:55 proteins
1:56 from the inside of the gel onto the
1:59 membrane
2:00 this gel membrane construct is flanked
2:03 on both sides
2:04 by filter papers all parts here are
2:07 soaked in buffer
2:09 to facilitate the electricity
2:10 transmission later
2:12 finally everything is fixed between the
2:15 cathode and the anode
2:17 the electric current pulls the
2:20 negatively charged proteins
2:22 which are located in the gel to the
2:24 positively charged a node
2:27 this transfers the proteins onto the
2:29 membrane
2:30 from now on the membrane has to be
2:33 incubated several times
2:35 the first step includes blocking
2:39 therefore the membrane is incubated with
2:41 milk
2:42 or bsa blocking should prevent
2:45 antibodies
2:46 from unspecifically binding to the
2:48 membrane or to other proteins
2:51 after blocking the membrane can be
2:53 incubated with the primary antibody
2:57 this antibody should be selected to only
3:00 bind to the protein of interest
3:03 after incubation with this primary
3:05 antibody a washing step will wash away
3:08 the unbound antibodies while not
3:11 removing the antibodies
3:12 that are bound to the protein of
3:14 interest the next step
3:16 involves the incubation with a secondary
3:19 antibody
3:19 which has to be specific for the primary
3:22 antibody
3:23 after washing only those secondary
3:26 antibodies remain
3:27 which are bound to the primary antibody
3:30 the detection of the protein
3:32 of interest is the last step in western
3:34 blood analysis
3:35 for that purpose the secondary
3:38 antibodies are conjugated to an
3:40 enzyme for many western blood assays
3:43 secondary antibodies are conjugated with
3:45 horseradish peroxidase
3:48 the protein of interest is bound by a
3:50 primary antibody
3:51 the secondary antibody binds to the
3:54 primary antibody
3:55 and when the membrane is incubated in a
3:57 substrate mixture
3:59 the enzyme converts the substrate
4:01 thereby producing a chemiluminescent
4:03 signal
4:04 that visualizes the protein band the
4:07 presence of the target protein in these
4:09 samples
4:10 has been confirmed make sure to
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