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In Topic: fatty acid free BSA-cell culture
31 January 2012 - 01:43 PM
BSA is used as a carrier of long- chain fatty acids. High concentration of free (unbound) FA are generally quite toxic for cells in culture.
In Topic: sublimation of frozen aliquots on storage
31 January 2012 - 12:13 PM
This is a well known problem in bio-banking. Usually it is best to use screw-caped tubes with rubber seals and store the tubes in small styrofoam boxes to minimize temperature shifts.
However, you are aliquoting very small volumes. Can you dilute say 20 fold in a buffer with a carrier protein and store 200 ul/tube as above? If you need to be carrier protein-free (for example for iodination) perhaps it would be better to freeze-dry the 10 ul aliquots.
However, you are aliquoting very small volumes. Can you dilute say 20 fold in a buffer with a carrier protein and store 200 ul/tube as above? If you need to be carrier protein-free (for example for iodination) perhaps it would be better to freeze-dry the 10 ul aliquots.
In Topic: sublimation of frozen aliquots on storage
31 January 2012 - 12:12 PM
This is a well known problem in bio-banking. Usually it is best to use screw-caped tubes with rubber seals and store the tubes in small styrofoam boxes to minimize temperature shifts.
However, you are aliquoting very small volumes. Can you dilute say 20 fold in a buffer with a carrier protein and store 200 ul/tube as above? If you need to be carrier protein-free (for example for iodination) perhaps it would be better to freeze-dry the 10 ul aliquots.
However, you are aliquoting very small volumes. Can you dilute say 20 fold in a buffer with a carrier protein and store 200 ul/tube as above? If you need to be carrier protein-free (for example for iodination) perhaps it would be better to freeze-dry the 10 ul aliquots.
In Topic: Problems with detergents and Ni-NTA
31 January 2012 - 11:58 AM
I have not come across the detergent you are using before (but it is obviously fairly non-denaturating). In this regard, are you sure that the tag is exposed in the partially denatured protein? To explore this possibility, maybe you could look at the effect of adding a denaturant (urea/guanidine) and see if this improves binding.
In Topic: Problem with soft agar assay
31 January 2012 - 11:41 AM
Changing the format may increase the time the bottom agar needs to FULLY set. Would suggest you try chilling the bottom layer at 4oC before plating the top layer. If problems persists maybe try a slight increase in agar concentration of the bottom layer (0.7%?).
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