| 5–12 |
Low yield with Golgi preparations |
Sucrose concentration not accurate Inaccurate handling of the experimental procedures
|
Check the sucrose concentration using a refractometer Take caution when handling the samples
|
| 5–12 and 60–73 |
Golgi stacked structure not preserved when examined by EM |
Over homogenization of the liver tissues Improper fixation for EM processing
|
Take caution when homogenizing the tissues and when handling the experimental procedures. Avoid the lipid contamination when taking the Golgi fractions from the first gradient. Prepare fresh fixative for EM processing
|
| 33–36 |
Golgi disassembly by mitotic cell cytosol does not work |
The quality of the mitotic cytosol is low Golgi membranes are more than 1 year old
|
Check whether the histone kinase activity in mitotic cytosol is at least 20× of that in interphase cytosol. Ensure that the mitotic index is above 95% and there is low number of apoptotic cells. Do not over crack the cells using the homogenizer. Avoid lipid contamination on the surface after the high-speed centrifugation. Prepare new Golgi membranes
|
| 42–44 |
Golgi reassembly by interphase cell cytosol does not work |
Disassembly reaction is not carried out properly The quality of the inter-phase cytosol is low
|
Ensure that the samples are properly handled in the disassembly reaction. Use low passage of HeLa S3 cells for cytosol preparation. Use thymidine or aphidicolin to block cells in the interphase. Add a kinase inhibitor into the reaction. Test liver cytosol. Ensure that no GTPγS is used in the disassembly reaction, it blocks membrane fusion
|
| 45–48 |
Golgi disassembly by purified proteins does not work, with a large number of long cisternae remaining |
ARF1 or coatomer preparations have low activity |
Ensure ARF1 is last added into the reaction. Increase the amount of ARF1 and coatomer in the reaction. Prepare new ARF1 and coatomer proteins
|
|
Golgi disassembly by purified proteins does not work, with the core of the Golgi remaining as mini-stacks |
Kinase activity is low |
Check whether the prepared cdc2 and plk kinases are active or make a new preparation. Increase the amount of the kinases. Include a phosphatase inhibitor into the reaction
|
| 49–53 |
Golgi reassembly by purified proteins does not work, with new mini-stacks generated but the cisternae are short and a large number of vesicles are remaining |
Disassembly reaction not done properly The quality of the purified proteins for membrane fusion is low
|
Ensure that the samples are properly handled in the disassembly reaction. Use interphase cytosol instead of purified proteins for disassembly as a positive control. Prepare new NSF and p97 and their adaptor proteins. Use gel filtration to remove the degradation products of the purified proteins
|
|
Golgi reassembly by purified proteins does not work, new cisternae are formed, but they do not form stacks |
Phosphatase (PP2A) activity too low |
Increase the PP2A amount in the reaction. Use a kinase inhibitor such as staurosporine. Prepare new phosphatases. Use interphase cytosol as a positive control. Use standard Golgi disassembly and reassembly as a positive control
|
| 54–59 |
No membranes in the vesicle fractions |
Gradients were not pooled accurately. COPI vesicles are uncoated Centrifugation did not reach equilibrium of the gradient
|
Ensure that the sucrose concentration is accurate and the gradients made properly. Run the gradient immediately after the reaction. Extend the centrifugation time
|
| 60–73 |
Under the EM, samples showed dark precipitates with less membranes after the standard disassembly and reassembly (this never happens with the defined assay) |
Proteins in the cytosol precipitated |
Spin the cytosol before the reaction to remove the precipitates. Spin the membranes through a 0.4 M sucrose cushion as given in Steps 37–41 after the reaction, then fix the membranes. Omit uranyl acetate stain of the EM sections
|
