Comments for Benchwise

Comment on Crystallography for beginners: making the move into protein crystallography – Part 2 by Chen Guttman

Chen Guttman — Sun, 21 Apr 2013 07:29:19 +0000

Thanks, Pro! Its great to get such good feedback!
Chen

Comment on Crystallography for beginners: making the move into protein crystallography – Part 2 by Chen Guttman

Chen Guttman — Sun, 21 Apr 2013 07:28:38 +0000

Hi Irish,
Yes, I indeed had found one post being completely copied and posted by a south-east Asian blogger (thief is much more accurate) but I had little to do about it. Part of publishing your blog online is the exposure for possible plagiarism. I wish wordpress could enforce a copy-block function such that only search bots could scan and extract the text, while keeping the text copyright protected from copy-pasting by users.
Unfortunantly, I am not aware of any function that can protect your text (unless you post it as an image but then the search bots can not scan that text so you’re loosing grounds here).
Have a great day and thanks for stopping by,
Chen

Comment on Crystallography for beginners: making the move into protein crystallography – Part 2 by project managment professional

project managment professional — Fri, 19 Apr 2013 01:01:43 +0000

I think the admin of this site is in fact working hard for his website, because here every stuff is quality
based information.

Comment on Crystallography for beginners: making the move into protein crystallography – Part 2 by Irish

Irish — Thu, 18 Apr 2013 05:58:51 +0000

With havin so much content and articles do you ever run into any issues of plagorism or copyright infringement?
My blog has a lot of exclusive content I’ve either written myself or outsourced but it appears a lot of it is popping it up all over the internet without my agreement. Do you know any methods to help prevent content from being ripped off? I’d genuinely appreciate it.

Comment on LinkedIn for graduate students: how to market yourself on the net by ryanbrown870

ryanbrown870 — Tue, 12 Mar 2013 19:33:39 +0000

This is something I have been thinking of doing for a while. I setup a LinkedIn account beforeI started my PhD but didn’t really use it much but will deffo give it another try

Comment on Purify, purify and…purify more: tips for improving your protein purification capabilities by Chen Guttman

Chen Guttman — Sat, 09 Feb 2013 13:39:16 +0000

Hi Cyrus,
I am performing WB in the same manner as I am doing WB from a non-stained gel – no special treatment or procedure.
Chen

Comment on Purify, purify and…purify more: tips for improving your protein purification capabilities by cyrus

cyrus — Fri, 08 Feb 2013 14:56:11 +0000

Hey, how do you western blot directly from your coomassie stained gel???

Comment on Purify, purify and…purify more: tips for improving your protein purification capabilities – part 5 by Chen Guttman

Chen Guttman — Thu, 13 Dec 2012 13:37:39 +0000

Yes, this can be a good strategy. Take into consideration that low abundant protein will be hard to observe under coomassie staining so you might want to concentrate the samples (irreversibly) through Trichloroacetic acid (TCA) - here's a protocol if you wish to explore. Good luck! Chen

Comment on Purify, purify and…purify more: tips for improving your protein purification capabilities – part 5 by Joe

Joe — Wed, 12 Dec 2012 20:35:21 +0000

Thanks so much for the great information, Chen! I’m actually interested in proteins across the molecular weight range–my intention is to separate the proteins out roughly by size and use the fractions for further experiments. I suspect there are proteins in the fractions where the absorbance is extremely low, as you suggest. Changing the scale does allow me to see more tiny peaks. Perhaps pooling a few runs after concentration should give me enough material for the lower abundance fractions.

Comment on Purify, purify and…purify more: tips for improving your protein purification capabilities – part 5 by Chen Guttman

Chen Guttman — Mon, 10 Dec 2012 05:00:29 +0000

Hi Joe,
After making some calculation I am taking back my statement about your working flow rate – it IS at the optimal range (which is 2-5 cm/hr). If you go below 2 cm/hr you risk diffusion of the peak (refer to Van Deemter equation).
In regard to diluting your sample – it really depends on the peaks height and width and since I can’t see the chromatogram its hard to comment on it. Generally speaking, injecting less material will inevitable lead to better resolution of peaks, but it will increase your work time by two fold on the one hand, and on the other you will dilute less prominent proteins which will make you identify and collecting them more difficult. You really need to see whether your TARGET protein is “overloaded”. Just look at the peak and observe if it is rounded at the top or flat. If it is flat than you’ve crossed the maximum reading of the inline spectrophotometer, which also means a LOT of protein on a 26/60 column.

One more point: when separating a sample in which there is a highly abundant protein and a low abundant protein, there is a chance that you will miss the low abundant protein altogether due to the wide scaling of the absorbance, especially on a large volume column such as the 26/60 series. If you’re suspecting that additional proteins are present in the sample (and you haven’t seen them in the previous runs), I suggest you use a Y axis scale of 200-500 mAU, even go as low as sub 100. This way you will notice most proteins which has some abundance in the sample.

And more importantly, if you’re looking to purify a low abundant protein in such a sample, you might want to consider salt/pH precipitation of the sample such you remove most of the abundant contaminants. Size exclusion chromatography excels when size differences are large and significant and makes a poor first step for highly contaminated samples.
Hope this helps!
Chen