No discussions found
Start your discussion
Share your thoughts or question with experts in your field
Start a discussion
Found 4 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
SDS-PAGE gels (8.5 or 12%) containing 25 µg of protein per well were transferred to 0.45
µm nitrocellulose (Bio-Rad 1620115) using constant voltage (100V) for 1 hr at 4°C in 1X
transfer buffer with 10% ethanol. After transferring, the nitrocellulose membrane was
blocked with 5% milk 1X TBST at room temperature for 30 minutes. The membranes were
then washed three times, five minutes each, with 1X TBST. |
Primary incubation with
antibodies was done overnight at 4°C. After primary incubation, the blots were washed
five times, five minutes each, with 1X TBST and then incubated in secondary antibody in
1% milk TBST for thirty minutes at room temperature. After secondary antibody
incubation, the blots were washed five times, five minutes each, with 1X TBST and then
once with 1X TBS for ten minutes. |
|
| Upstream tips |
SDS-PAGE gels (8.5 or 12%) containing 25 µg of protein per well were transferred to 0.45
µm nitrocellulose (Bio-Rad 1620115) using constant voltage (100V) for 1 hr at 4°C in 1X
transfer buffer with 10% ethanol. After transferring, the nitrocellulose membrane was
blocked with 5% milk 1X TBST at room temperature for 30 minutes. The membranes were
then washed three times, five minutes each, with 1X TBST. |
| Protocol tips |
Primary incubation with
antibodies was done overnight at 4°C. After primary incubation, the blots were washed
five times, five minutes each, with 1X TBST and then incubated in secondary antibody in
1% milk TBST for thirty minutes at room temperature. After secondary antibody
incubation, the blots were washed five times, five minutes each, with 1X TBST and then
once with 1X TBS for ten minutes. |
| Upstream tips |
Protocol tips |
Downstream tips |
SDS-PAGE gels (8.5 or 12%) containing 25 µg of protein per well were transferred to 0.45
µm nitrocellulose (Bio-Rad 1620115) using constant voltage (100V) for 1 hr at 4°C in 1X
transfer buffer with 10% ethanol. After transferring, the nitrocellulose membrane was
blocked with 5% milk 1X TBST at room temperature for 30 minutes. The membranes were
then washed three times, five minutes each, with 1X TBST. |
Primary incubation with
antibodies was done overnight at 4°C. After primary incubation, the blots were washed
five times, five minutes each, with 1X TBST and then incubated in secondary antibody in
1% milk TBST for thirty minutes at room temperature. After secondary antibody
incubation, the blots were washed five times, five minutes each, with 1X TBST and then
once with 1X TBS for ten minutes. |
|
| Upstream tips |
SDS-PAGE gels (8.5 or 12%) containing 25 µg of protein per well were transferred to 0.45
µm nitrocellulose (Bio-Rad 1620115) using constant voltage (100V) for 1 hr at 4°C in 1X
transfer buffer with 10% ethanol. After transferring, the nitrocellulose membrane was
blocked with 5% milk 1X TBST at room temperature for 30 minutes. The membranes were
then washed three times, five minutes each, with 1X TBST. |
| Protocol tips |
Primary incubation with
antibodies was done overnight at 4°C. After primary incubation, the blots were washed
five times, five minutes each, with 1X TBST and then incubated in secondary antibody in
1% milk TBST for thirty minutes at room temperature. After secondary antibody
incubation, the blots were washed five times, five minutes each, with 1X TBST and then
once with 1X TBS for ten minutes. |
| Upstream tips |
Protocol tips |
Downstream tips |
| Cells were lysed in RIPA buffer with PMSF (PMSF:RIPA=1:100) for 30 min on ice and then centrifuged for 15 min. Protein concentration of the supernatant was measured using a BCA protein assay kit. |
Equal amounts of protein from cell extracts were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a PVDF (polyvinylidene fluoride) membrane (30). The membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies conjugated with horseradish peroxidase. |
Finally, the protein level was determined using a chemiluminescent approach in a dark room. |
| Upstream tips |
| Cells were lysed in RIPA buffer with PMSF (PMSF:RIPA=1:100) for 30 min on ice and then centrifuged for 15 min. Protein concentration of the supernatant was measured using a BCA protein assay kit. |
| Protocol tips |
| Equal amounts of protein from cell extracts were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a PVDF (polyvinylidene fluoride) membrane (30). The membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies conjugated with horseradish peroxidase. |
| Downstream tips |
| Finally, the protein level was determined using a chemiluminescent approach in a dark room. |
| Upstream tips |
Protocol tips |
Downstream tips |
| Cells were lysed in RIPA buffer with PMSF (PMSF:RIPA=1:100) for 30 min on ice and then centrifuged for 15 min. Protein concentration of the supernatant was measured using a BCA protein assay kit. |
Equal amounts of protein from cell extracts were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a PVDF (polyvinylidene fluoride) membrane (30). The membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies conjugated with horseradish peroxidase. |
Finally, the protein level was determined using a chemiluminescent approach in a dark room. |
| Upstream tips |
| Cells were lysed in RIPA buffer with PMSF (PMSF:RIPA=1:100) for 30 min on ice and then centrifuged for 15 min. Protein concentration of the supernatant was measured using a BCA protein assay kit. |
| Protocol tips |
| Equal amounts of protein from cell extracts were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a PVDF (polyvinylidene fluoride) membrane (30). The membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies conjugated with horseradish peroxidase. |
| Downstream tips |
| Finally, the protein level was determined using a chemiluminescent approach in a dark room. |
Can't find the product you've used to perform this experiment? It would be great if you can help us by
Adding a product!