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Yes, you can use SDS-PAGE as a semiquantitative estimate of protein concentration. You need to create a standard curve with a protein of known concentration to compare against. Quantification is done by densitometry. It's a quick and easy process, but keep in mind some limitations: Band intensity depends not only on the amount of protein but also on the ...


5

This is a classical protein purification problem - you have to find ways to fractionate your mixture so that each fraction can be assayed for the activity you are interested in. When you find the active fraction you then subject that to a different type of fractionation. Salting out The solubility of proteins is affected by the ionic strength of the buffer....


5

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 interaction of aromatic aminoacids with the UV light. If you have a protein with few or no aromatic amino acids, your measurement will be wrong. The same is ...


4

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 value to give you an accurate concentration reading. To find the extinction coefficient for your protein, put its whole sequence into this server http://web....


3

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 involves substituting uridine (U) with 4-thiouridine (4SU) or substituting guanosine (G) with 6-thioguanosine (6SG). The rationale behind using these modified ...


3

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 sequence, then A-280 measurement is a very accurate, fast, nondestructive method of determining protein concentration. It is certainly much more reliable than any ...


3

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 of Sephadex G-75 is about 7.5ml. This value refers only to the water contained within the gel particles and not to the water trapped outside of the particles ...


2

If you don't want acrylamide in your preparation, don't use it, as you will always have some carry-over in the solution. And you will not get rid of it completely, as it at least partly co-precipitated with the nucleic acids (it's used as a co-precipitation agent for this purpose). I think the best solution is to use chromatography, either size-exclusion ...


2

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 detected immunologically. The small size of the epitope reduces the chance it will disrupt the protein's normal function (to which it's tagged). If we look at ...


2

A longer or duplexed Nucleic acid will have more charge per mole of the molecule. But generally it is not the overall charge that matters in electrophoresis or MS or chromatography; it is the charge density and specific charge (z/m) that matters (charge density of a longer RNA will be almost same as smaller RNA. they will also have same specific charge) The ...


2

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 concentration, there is an initial large influx of water which can stretch the tubing. If this is repeated several times you may stretch the pores to where they no ...


2

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 solution and the protein is well characterised. Entering the correct extinction coefficient will improve the accuracy of the concentration calculation. If using ...


2

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 below. The manuals for your ÄKTA system and the HisTrap columns discuss pressure in some detail as well. How can backpressure be created in a system that is not ...


2

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 allow proteins up to 200kd to pass through. There are some proteins that are larger. Editing with some source material. Nice Presentation on Pore Sizes and ...


2

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 from the rest of the cell is mostly dependent on the rotor of the centrifuge, because both together determine the acceleration you get, which should be in the ...


2

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 chromatography and use their procedure as a starting point. Some generalities to keep in mind for buffer selection: Buffers are most effective when the desired pH is ...


2

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 Permeation Chromatography is compromised by the close proximity of Lysozyme (14.3 KDa) and Cystatin (12.0 KDA) in their molecular mass. Sephadax G100 separates a ...


2

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 Sephadex, but they also reference other methods in the introduction that you could take a look at. Both of the methods you described sound reasonable. The only ...


1

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 exactly. If you have a surface like a lab bench, you use ethanol or iso-propanol because its cheap, quick, relatively safe to use for that purpose and dries up ...


1

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 to the column. See for example the protocol for the Monarch® PCR & DNA Cleanup Kit. Consequently I would recommend just doing an ethanol precipitation, ...


1

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% Isopropanol 1M NaOH 1M NaCL SDS in 0.1M Acetic Acid Stripping buffer (EDTA) Wash between each with several column volumes of distilled water. You may not need to do ...


1

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 be found here. I did a very quick and dirty comparison of the frequency that human ACSL_1 (Uniprot ID; P33121) and GAPDH were detected in control purifications. ...


1

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 lawn). Each plaque should be the progeny of one original phage, although sometimes two or more phages could be very close to each other on the plate and it will ...


1

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 down the imidazole concentration at which your protein comes off the column, you can in future just stick to concentrations around that area, though it's still ...


1

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 fusions. That said, do you have a reason for inserting the the tag after methionine? The T7 tag already starts with methionine and naturally functions to ...


1

The standard when purifying GST with Glutathione agarose beads is to use STE buffer (10 mM Tris-HCl (pH 7-7.5), 150 mM NaCl and 1 mM EDTA) for washes and we have had no problem with this in our lab!


1

It works pretty well and can be used to desalt DNA. The DNA runs a bit different than proteins since it is more a long stretched molecule while a lot of proteins are globular. See this references (the first is about the behaviour of DNA on superose): Size-exclusion chromatography of DNA restriction fragments. Fragment length determinations and a comparison ...


1

The pI (iso-electric point) is the pH at which the protein (or other molecule), overall has a net zero charge. As @Chris points out, the buffer you are using will change the pH the protein finds itself in. This will change the charge of the protein. In ion exchange chromatography, there are charged chemical groups on the resin and these can cause a ...


1

Have a look at this table, it really depends on the buffers you use: The table is from this website, which is definitely worth reading when you are interested (or work) with ion exchange chromatography.


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