Find content
Male
native protein will have the correct conformation, subunit structure, charge, size, etc.
denatured protein will give you subunit size.
some antibodies will only recognize denatured protein, some will only recognize native protein, and some will recognize both.
#154847 What is different between Native protein and Denatured protein samples.
Posted
on 09 May 2013 - 04:54 AM
#139334 HPRT mutation & 6-TG selection assay
Posted
on 12 August 2012 - 01:15 PM
Cells that survive 6-TG selection should form colonies, not exist as single cells. The colonies appear like the colonies you would see in antibiotics selection to establish stable cell line. A typical colony looks like the one in the image (d and f). Single cells are just dying cells which have been poisoned by 6-tg.
#142323 question about protein expression normalization.
Posted
on 26 September 2012 - 07:12 PM
Total protein is only as accurate as the assay you use to measure it. Having done westerns for quite a few years now, I would say that the accuracy of most protein content assays is somewhat questionable due to the chemistry of the assay. For instance in the BCA assay (one of the more reliable) the values obtained for different proteins depend on the number of cys-cys, tyr and trp residues in the protein... if you use BSA as a standard, your total protein values are not accurate (though they might be precise)! Then you have to ensure that your samples lie within the reference range and that you are not exceeding the capabilities of the sectrophotometer used to read the assay, as well as ensuring that you have used the appropriate standard curve.
Quantitation itself is problematic as it depends heavily on correct exposure of the film, and a number of variables during the blotting process such as even transfer at all MW, affinity of the antibodies for the different proteins.
You might be better off normalizing to bands from a ponceau stain of the membrane.
Quantitation itself is problematic as it depends heavily on correct exposure of the film, and a number of variables during the blotting process such as even transfer at all MW, affinity of the antibodies for the different proteins.
You might be better off normalizing to bands from a ponceau stain of the membrane.
#138557 HPRT mutation & 6-TG selection assay
Posted
on 30 July 2012 - 07:47 AM
The concentration you used appears a bit too high (we use 5 ug/ml for our cell line). You have to do a killing curve using the cell line you are working with and then use the lowest dose that kill all your wildtype cells for selection.
Some protocols recommend that your wait for 8 days after introducing mutation before adding 6-TG.
you may also need to treat your cells with MNNG as positive control which is know to cause HPRT mutation and gives colonies on 6TG selection.
Here is a nice protocol on HPRT mutation assay
HPRT_protocols_042009.pdf 58.62K
161 downloads found at http://depts.washing...cols_042009.pdf
this paper also gives a protocol" Frequency of HPRT gene mutations induced by N-methyl-N'-nitro-N
http://www.ncbi.nlm..../pubmed/9626969
Some protocols recommend that your wait for 8 days after introducing mutation before adding 6-TG.
you may also need to treat your cells with MNNG as positive control which is know to cause HPRT mutation and gives colonies on 6TG selection.
Here is a nice protocol on HPRT mutation assay
HPRT_protocols_042009.pdf 58.62K
161 downloads found at http://depts.washing...cols_042009.pdfthis paper also gives a protocol" Frequency of HPRT gene mutations induced by N-methyl-N'-nitro-N
http://www.ncbi.nlm..../pubmed/9626969
