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- Active Posts 225
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- Age 31 years old
- Birthday May 21, 1982
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Male
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Location
Cambridge
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My research interests
immunology, molecular biology, biochemistry
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In Topic: TAE Vs TBE buffer
11 September 2012 - 02:37 AM
As leelee said TBE causes less heating because borate ions have better mobility than acetate. The problem with using borate is if you want to excise your band out of the gel and purify it the borate ions form complexes with the DNA and the yield is much lower. Therefore people use TAE. There was a paper on this subject a while ago called "Not your father's buffer" and it said that the best is Li-borate or Na-borate and in my experience you can really speed things up with a different buffer.
In Topic: TCA Precipitation to remove albumin
21 August 2012 - 02:27 AM
There are a number of products on the market. Why don't you just do a Google search for a kit and try to find something that's within your budget. I know of kits from Qiagen and GE, but I don't know if they are within your price range.
In Topic: TCA Precipitation to remove albumin
19 August 2012 - 11:51 PM
It it were me, I would not use TCA to remove albumin, because it non-selectively precipitates all proteins. There are kits available for albumin depletion, which are used prior to 2D electrophoresis and I have heard they're quite OK.
As for dissolution of your pellet, I would go with SDS buffer and BCA or Bradford for quantification.
As for dissolution of your pellet, I would go with SDS buffer and BCA or Bradford for quantification.
In Topic: Biotin Avidin based Competition ELISA
26 June 2012 - 10:07 PM
Can you elaborate more on what type of ELISA you are performing? What you are coating, what you are detecting and what level of signal you are getting...
In Topic: F(ab’)2 Preparation method??
29 May 2012 - 03:28 AM
In theory you can generate the F(ab')2 fragment with pepsin, although I don't have direct experience with that. In our hands ficin did not work very well.
I have done many Fab preparations, digesting the IgG1 with papain. For purification the best way is to remove the undigested Abs with protein A or G and then maybe do another purification step - I use an affinity column that I prepared myself with anti-Fab antibodies.
I have done many Fab preparations, digesting the IgG1 with papain. For purification the best way is to remove the undigested Abs with protein A or G and then maybe do another purification step - I use an affinity column that I prepared myself with anti-Fab antibodies.
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