2-D Clean-Up Kit
The 2-D Clean-Up Kit is designed to prepare samples for 2-D electrophoresis that otherwise produce poor 2-D results due to high conductivity, high levels of interfering substances or low concentration
FAQ
Is there anything that 2-D Clean-up kit is not compatible with?
Results may be less than impressive with samples that contain high concentrations of SDS or guanidine. Samples with more than 2% SDS or 1 M guanidine should be avoided (or diluted prior to application of the kit).
What is the difference between the 2-D Clean-Up Kit and the SDS-PAGE Clean-Up Kit?
Four of the reagents (Precipitant, Co-precipitant, Wash buffer and Wash additive) are identical. The SDS-PAGE Clean-Up Kit has three additional reagents (Buffer I, Buffer II and SDS-PAGE sample buffer). The additional reagents are for optimal solubilization in a small volume of SDS-PAGE-compatible sample buffer.
When should I use 2-D Clean-up kit?
2-D Clean-up kit is intended for "problem" samples that are either dilute, or have high levels of interfering substances that prevent effective focusing, or cause staining problems. If a sample focuses poorly, or there is a lot of background staining, application of this kit should be recommended.
Which 2-D gel thickness should I use?
Either 1.0- or 1.5-mm-thick spacers can be used for all vertical formats. Thinner gels stain and destain more quickly and generally give less background staining. Thicker gels allow easier positioning of the IPG strip on the surface of the SDS gel and have higher protein capacity. Thicker gels are also less fragile and easier to handle.
How does the use of 2-D Clean-up kit influence 2-D spot pattern?
No instances of spot positions changing due to application of 2-D Clean-up kit have been observed, only rare and isolated instances of specific spot losses. In many cases, spot count increases because the spot resolution has become sharper.
How do I prepare immunoprecipitated proteins for DIGE labeling?
Immuno precipitated proteins can be removed from the beads at low pH with 100 mM glycine-HCl, pH 2.7. After neutralization with Tris base the sample can be desalted using the 2-D Cleanup kit. The sample is now ready for CyDIGE labeling using the standard protocol.
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
2-D clean-up-kit
Symptom: There are disturbances at the ends of the IPG strip or horizontal streaking in the subsequent 2D gel. Current is high or voltage is low during first dimension IEF.
| Possible cause | Suggested remedy |
|---|---|
Insufficient washing with wash buffer. |
Ensure that the pellets is thoroughly dispersed and vortex frequently during incubation with wash buffer. |
Contaminated additives. |
Check the quality of the additives used for solubility. Please note that urea can degrade easily to charge products. |
Addition of salts during cell preparation. |
Avoid spinning down cells in phosphate-buffered saline (PBS). Do wash the cells, however, because cell culture medium is also isotonic. But instead of using PBS for washing use salt-free buffer/osmoticum, such as 10 mM Tris, 250 mM sucrose, pH 7.0. |