7
votes
Accepted
Measuring protein concentration, Bradford vs. Nanodrop?
That depends strongly on your protein and how exact you need this concentration. Both tests (Bradford and the measurement at 280nm) only do an approximation.
The measurement at 280nm relies on the ...
- 51.1k
6
votes
Measuring protein concentration, Bradford vs. Nanodrop?
The nanodrop should have an option that allows you to input an "extinction coefficient". This is a measurement of how much one mole of your protein will absorb at 280nm. The nanodrop will use this ...
- 71
5
votes
What's the best way to purify my His tagged protein? Supernatant super viscous after first sonication?
As Chris said in the comments, make sure you have sufficient DNase in your lysis buffer. That combined with good sonication will definitely shear nucleic acids and make your solution less viscous. If ...
- 15.2k
5
votes
Why are IMAC and gel filtration combined?
Without knowing your specific protocol, it is common to use more than one purification method for a single protein. Given the broad similarities that all proteins and even most biomolecules share, it ...
- 17.6k
3
votes
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Why does 4-thiouracil labelling to map RNA-binding proteins cause a T-C change?
The technique used in that paper is called PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation; see Ascano et al., 2012 and Spitzer et al, 2014). This technique ...
- 35.2k
3
votes
Sephadex column for alpha-amylase
The '75' of Sephadex G-75 refers to the water regain value, which is defined as the grams of water absorbed upon hydration of 1g dry powder, multiplied by 10 (see Reiland). Thus the water-regain value ...
- 4,658
3
votes
Measuring protein concentration, Bradford vs. Nanodrop?
If you are dealing with a pure protein where there is nothing else present that will absorb at 280nm and if the E(1%, 280) of the pure protein is known or may be calculated from the amino acid ...
- 4,658
3
votes
Accepted
Will dissolved proteins pass through a 0.2 micron filter?
Many proteins will pass through a 0.2 micron filter. If the proteins aggregate or if they stick to the filter material because it is charged, they may not.
Barring the above, a 0.2 micron filter will ...
- 1,309
2
votes
Accepted
Clarify pre-column pressure vs. system pressure vs. backpressure for prepacked columns used with the äkta purification system
Is there a good article or webpage that goes through this theory at a basic level?
Yes, Chapter 7 of the manual linked to by Andreas Tosstorff is a good source, and is also the source of the images ...
- 17.6k
2
votes
T7 Tagging in Synthetic Biology
The T7 tag is the first 11 amino acids of the T7 gene 10 protein. Basically, if you engineer a protein with the T7 tag, you've engineered a small, known epitope to the protein of interest which may be ...
- 8,081
2
votes
Measuring protein concentration, Bradford vs. Nanodrop?
The choice between colorimetric and direct quantification at A280 depends on both the protein to be quantified and the buffer being used. Generally A280 works well when you have a purified protein ...
- 21
2
votes
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Can one use dialysis tubing several times?
This is more of a long comment than an answer, since I don't know. It could be okay, but if you are unsure whether it will work, is it really worth the risk? Here are some thoughts:
With high AS ...
- 17.6k
2
votes
Ion-exchange vs Gel Permeation Chromatography for proteins
Here is what I have come up with. Thank you for all your help.
Ion Exchange Chromatography(IEC) will yield the purist product from a single application. The main reason is due to the fact that Gel ...
- 43
2
votes
Accepted
Ion-exchange vs Gel Permeation Chromatography for proteins
Since lysozyme is so well studied, your best bet is to search the literature and see what methods other people use. See this paper, for example. They report that lysozyme can be affinity purified on ...
- 17.6k
2
votes
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Centrifuge after sonication
No, if you use only centrifugation to separate proteins from other parts of the (bacterial) cell, you need very high centrifugal forces. The actual speed (in rpm) you need to separate soluble proteins ...
- 4,356
2
votes
What buffer should I choose for IEX chromatography for purifying IgY
Your column manual will probably come with buffer recommendations (these manuals are also often available online). You can also search for publications that have purified IgY by anion exchange ...
- 17.6k
1
vote
Which is more efficient as a germicide: UVC, Ozone or combination and why?
Your question contains the word "efficient". The most efficient method is the one with achieves the goal with the minimum of effort or disadvantages. And that totally depends on what you want to do ...
- 382
1
vote
Separate DNA fragments with very different size (oligo and lambda-DNA)
A spin column might work for your purposes since you can do multiple rounds of binding if your volume is too large to bind in a single pass, but for DNA of that size a lot will be irreversibly stuck ...
- 5,513
1
vote
Ligation without purifying insert
Thanks all for your comments guys. I tried purifying using PCR purification kit. I could recover 120 ng of purified fragment from 500 ng. It is ok for my further work.
- 630
1
vote
False positives in TAP - MS experiments
The common repository for affinity purification (CRAPome) contains the results from many tandem affinity experiments and scores them based on their presence in control purifications.
The database can ...
- 1,308
1
vote
Methods for phage separation
Look up "phage purification." Basically, you add your phage solution on top of your host bacteria on a plate and incubate. As the bacteria grow, you should see plaques (clear areas in the bacterial ...
- 11
1
vote
Dissolving cell pellet after sonication
Follow the instructions that came with your column for cleaning. The resin will probably be fine, but you'll need to thoroughly clean the column. My GE columns recommend washes of:
20% EtOH
30% ...
- 1,301
1
vote
Accepted
Protein purification: Linear elutions or stepwise elution, using imidazole or pH gradient?
Linear is better typically. The flow rate combined with fraction size and length of time gives you better resolution for which proteins are coming off the column at what point.
Once you've narrowed ...
- 1,301
1
vote
T7 Tagging Next to Met
By "still work" I assume you are asking if it will still be recognized by an antibody? It might. Internal T7 tag fusions are done, though they may require different antibodies than terminally tagged ...
- 17.6k
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