By Steven D. Schwartzbach, Omar Skalli, Thomas Schikorski
This quantity provides authoritative and state of the art tools and protocols targeting 3 software containers masking the more and more various methodologies used to snapshot chosen proteins and to enquire their functionality by means of gentle and electron microscopy. the 1st software field comprises the improvement of quite a lot of molecular and immunological probes to focus on particular proteins. the second one info using those probes for top solution fluorescence microscopy and the 3rd specializes in purposes for transmission and scanning electron microscopy. Written within the hugely winning Methods in Molecular Biology series layout, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, effectively reproducible laboratory protocols, and tips about troubleshooting and averting recognized pitfalls hence making sure profitable leads to the extra research of this important field.
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Additional info for High-Resolution Imaging of Cellular Proteins: Methods and Protocols
May result in shearing of cells. Coverslips should never be aspirated to the point of being completely dry, as this will lead to alterations in cellular morphology. 29. Some antibodies may require fixation in methanol. In these cases, cells should be incubated in methanol at −20 °C for 5 min, followed by 5–6 washes with PBS. Methanol causes both fixation and cell permeabilization, so steps 5 and 6 may be eliminated when using this method. 30. GFP-tagged proteins can be visualized without the use of antibodies, although antibodies may enhance signal.
4. Block the unoccupied sites with 300 μL/well of blocking solution. 5. Wash the wells three times with PBST. 6. Prepare serial dilutions of antiserum with PBST ranging from 1:300 to 1:300,000. 7. Add 300 μL/well of the antiserum serial dilutions to the wells and incubate for 2 h at 37 °C. 8. Wash the wells three times with PBST. 9. Dilute the secondary antibody 1:7000 with PBST. 10. Add 300 μL/well of the secondary antibody to the wells and incubate at 37 °C for 2 h. 11. Wash the wells three times with PBST.
5 μL for 24-well plate) (see Note 17). 5 μg for a 24-well plate). The total volume of the plasmid DNA should be less than 20 μL (10 μL for a 24-well plate). 11. Incubate the two solutions for 5 min at room temperature. 12. Following the incubation, combine the two solutions and mix by pipetting or inversion. Incubate mixture for 20 min at room temperature to allow the DNA and transfection reagent to complex. The mixed solutions should not be incubated for longer than 45 min before addition to cells.