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Thank you guys
I will try to use that soon
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In Topic: SH-SY5Y and Poly-L-Lysine
Yesterday, 08:14 AM
In Topic: protein concentration and Pull down
04 January 2013 - 02:34 PM
So you mean I could use 10ug or 100ug of bait protein and obtain the same result (it sound a little bit weird for me though ) ,
and what about the cell extract ?
I don't think that in this case, using a concentration of protein either 1mg/ml or 8mg/ml the result is the same
it's true that there are a lot of variabilities like the affinity of my protein with the bait, if it's just one protein to pull down or more then one.
Probably the best way could be to make a test with different concentrations of cell extract protein
and what about the cell extract ?
I don't think that in this case, using a concentration of protein either 1mg/ml or 8mg/ml the result is the same
it's true that there are a lot of variabilities like the affinity of my protein with the bait, if it's just one protein to pull down or more then one.
Probably the best way could be to make a test with different concentrations of cell extract protein
In Topic: Mass Spec software
29 December 2012 - 08:52 AM
Thank you Bob1, OpenChrom seems a good one
In Topic: dialyse of a protein
18 December 2012 - 04:27 PM
I use to snap freeze the cells after the induction as well, otherwise the experiment would be too long just in one day.
I tried as I mentioned before to use the filter tubes with a MWCO of 30 kDa, finally the protein is pure, but the yield is about the 25%,
I am sure that changing something during the purification I can still have more protein.
Thank you hoyajm, I am new working with proteins and for sure I will have some more question in the future
have good holidays
-P
I tried as I mentioned before to use the filter tubes with a MWCO of 30 kDa, finally the protein is pure, but the yield is about the 25%,
I am sure that changing something during the purification I can still have more protein.
Thank you hoyajm, I am new working with proteins and for sure I will have some more question in the future
have good holidays
-P
In Topic: dialyse of a protein
12 December 2012 - 01:57 PM
Thank you for your answer Enthusiast,
I will try soon and Il l let you know what happened, I tried to change different conditions such as different pH for the binding and washing, different concentration of imidazole either for the binding or for the washing but the results was to have less protein and less contaminants.
May I ask you what do you usually use for the cell lysis ?
what I do is to follow their protocol , plus protease inhibitor (PMSF) in the lysis buffer.
I don't use lysozyme and I go throw the sonication
I will try soon and Il l let you know what happened, I tried to change different conditions such as different pH for the binding and washing, different concentration of imidazole either for the binding or for the washing but the results was to have less protein and less contaminants.
May I ask you what do you usually use for the cell lysis ?
what I do is to follow their protocol , plus protease inhibitor (PMSF) in the lysis buffer.
I don't use lysozyme and I go throw the sonication
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