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PhDinAcronyms

Member Since 01 Jun 2010
Offline Last Active May 13 2013 09:48 PM

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Active Posts 33
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Female
Location
Sydney

About me

Real Name
Kristina
Institution
Lowy Cancer Research Centre, UNSW
My research interests
Disulfide bonds, thiol/cysteine chemistry, thioredoxin, coagulation, angiogenesis, proteases, small molecule/protein-protein interactions

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bigudukaz
08 Apr 2013 - 04:09
Harvardstudent
03 Jun 2010 - 19:10

Posts I've Made

In Topic: Do cell signaling antibodies work with 1h RT incubation?

07 April 2013 - 08:28 PM

It will depend on how much of your protein is in the sample and the antibody but the short answer is yes it can work. I've probed for lots of different proteins (including some other cell signaling one) using 1 hr incubation with the primary and 1 hour with the secondary at room temp. It usually works, otherwise overnight can give a stronger signal.

In Topic: Cleavage of GST-tag with Factor Xa

14 March 2013 - 04:15 PM

Does your protein show up on SDS-PAGE before cleaving with FXa? If it does and then disappears after adding FXa, it's possible FXa could be cleaving your protein at additional sites, check the sequence to see if there might be other possible cleavage sites.

Also, GST is a pretty big tag (~26kDa), so will change the size of your protein significantly, so make sure your SDS-PAGE conditions are optimal for the cleaved size protein and it's not running off the gel. You should see both the cleaved protein and the GST tag though.

In Topic: Long term storage for RNA extracted from pancreas

03 March 2013 - 02:26 PM

There are commercial solutions- I have used RNAlater which preserves the RNA in tissue and cells and there is a solution that apparently preserves isolated RNA but I've never tried it:
http://www.biomatric...m/rnastable.php I'm not sure what is in it but might be useful.

In Topic: Using centrifugal filter units to concentrate protein

28 February 2013 - 06:36 PM

Under the specifications tab on the webpage it gives these speeds: Swinging bucket rotor: 4,000 x g; Fixed angle rotor (35°): 5,000 x g

I reuse them several times but within the same day and for the same sample. You don't want the membrane to dry out between each use either.

In Topic: Recommendation for GOOD anti-6xHis-tag and anti-Strep-II-tag antibodies?

14 February 2013 - 04:01 PM

Qiagen also has a variety of anti-his antibodies that can be specific to your his tag if you don't have a simple 6xhis that is easily detected (5xhis, 4xhis etc) and they also have nickel agarose for purification. You can buy it all as a kit

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