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5 votes

How to find out what biological effects a molecule has, without having a specific mechanism or pathway in mind?

Personally I think this is a poor question, because the answer is immediately obvious to anyone who was worked with current technologies for both proteins and nucleic acids, but as always, it is hard ...
bob1's user avatar
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5 votes

Decreasing signals in assay measurements

Freeze-thaw cycles are often suspected of causing degradation in organic molecules 1,2. My first guess would be that your fluorophore is breaking down due to those repeated cycles. Alternatively, you ...
tyersome's user avatar
  • 5,598
4 votes
Accepted

How can enzymes be immobilised on glass?

Glass can be functionalized by organosilanization so that biomolecules can be covalently attached via some cross-linker. As an example, one might aminosilanize the glass and then cross-link their ...
canadianer's user avatar
  • 17.8k
4 votes

Why such strange enzyme kinetics?

Worth considering whether it is an effect of the instrument or the system. What would happen if you preincubated your plate for 10 minutes? Would you have the "dip" after 25 minutes or again ...
Bjarte Lund's user avatar
3 votes

Improving contrast between dot and paper in dot blot

In the absence of another answer, I'll transition my comments to an answer. Please label your images. Both an Abcam guide and a Thermo troubleshooting document suggest using a kit or DAB protocol ...
Maximilian Press's user avatar
3 votes
Accepted

How much effort is it to establish a cytotox assay for cancer cell lines against a small number of possible compounds?

The answer to this is, of course, "it depends". There are quite a few factors at play here. As you don't seem to be experienced at cell culture yet, I strongly recommend that you find ...
bob1's user avatar
  • 12.5k
3 votes

Can ELISA be used to detect a plant enzyme? Creating assay for a new enzyme

I want to add something on top of Chris answer. The production of an antibody it is usually a quite slow (and expensive) process, an alternative that worth to consider is phage display (https://en....
alec_djinn's user avatar
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3 votes
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Using autoclaved store-bought distilled water for labwork?

Yes, distilled water would be absolutely fine for your needs. You really don't need ultrapure water except for the most sensitive of applications. You also don't need to autoclave the distilled water ...
canadianer's user avatar
  • 17.8k
2 votes

Why would you plate cells at different densities in a colormetric assay?

The comments you have given are some reasons, although they also depend on the confluency of the cells. Your teacher/professor/advisor is also making sure that you can see an effect at low levels of ...
MattDMo's user avatar
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2 votes
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Where can I find hemagglutination inhibition (HI) assay data?

I don't think there's any universal database containing ongoing, widely representative HI assay output. The Antigenic Cartography group has made available some historical datasets that were used in ...
iayork's user avatar
  • 14.3k
2 votes

Can ELISA be used to detect a plant enzyme? Creating assay for a new enzyme

I think the most promising routes use antibodies. You could either develop an ELISA or do western blot analysis of plant material - both need a good and specific antibody. To generate these, the ...
Chris's user avatar
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2 votes

TMB vs ECL for ELISA: which detection method is more sensitive?

In general, ECL is more sensitive than TMB for ELISA detection. The minimum detection limit of TMB is 60pg/mL and 20pg/mL for ultra TMB [1], whereas the detection limit of ECL can be as low as about 1....
Leading Biology's user avatar
2 votes

Is there a biological or technical reason for a 40 Ct cutoff in nucleic acid detection assay?

This is done because at some point you start amplifying nonsense and not get a meaningful signal. If (and this is most often not the case) you have the ideal doubling of you DNA template in each cycle,...
Chris's user avatar
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2 votes
Accepted

Cell viability assay: Problems with MTT assay in the solubilization step

The easy answer is to use acidified SDS 10 Percent in 0,01 N HCl. I added this directly, to my media and did not have any problems with crystal removal anymore. The acid will remove the coloring of ...
raptorlane's user avatar
1 vote
Accepted

Can I have LAMP on the bench / in the field?

I don't do chemistry or bench work, so I can't speak to conditions required for LAMP in the lab. However, I know people who have developed a portable LAMP set-up that works in the field. It's still ...
John Polo's user avatar
  • 136
1 vote
Accepted

What is the dynamic range under initial conditions?

I think that they mean initial profiling as the profiling/screening is the subject of the clause. In this case it would mean that FRET with these compounds has enough difference between cleaved and ...
bob1's user avatar
  • 12.5k
1 vote

Antibody detection - infection-induced vs vaccine-induced - is it common (or not) for a test to be positive for both?

Short answer: no. Because all vaccines (that I know of) rely on protein or polysaccharide production in some form, whether "natural" (e.g. inactivated virion, subunit vaccines) or "...
bob1's user avatar
  • 12.5k
1 vote

Enzyme inhibitor leads to higher turnover rate?

While it's difficult to know just what is going wrong in somebody else's laboratory, apparently paradoxical effects like these are unfortunately common in laboratory work. Assuming that these are ...
jakebeal's user avatar
  • 7,007
1 vote

Why is thymidine incorporation (DPM) normalized to per mg protein content while testing for cell proliferation?

I'm not sure that there is a strongly disciplined reason for the mg protein content as the normalization, but there is obviously a need for some normalization for different amounts of tissue- if you ...
Maximilian Press's user avatar
1 vote

Finding total concentration of enzymes

Hi again TheLast Cipher, Enzymes in a cell are usually semi-quantified by the: Western Blot technique. But there are lesser known techniques. E.g. For a Western Blot the unit of measurement is the ...
Andrew's user avatar
  • 714
1 vote

Easy and cheap antigen/antibody couple

A good choice is the IgG/anti-IgG pair for cheap, easy to demonstrate/work with system. E.g. Goat-IgG coupled with donkey anti-goat IgG. Just about any antibody supplier will have these. These type ...
Bob Tomas's user avatar
1 vote
Accepted

Can experiments in ELISA kits be monitored via both fluorometry and photometry?

can we use both methods depending on whether the marker enzyme on the antibody is given a fluorescent or chromogenic substrate? This is essentially correct. You can have the same ELISA work on ...
CKM's user avatar
  • 8,119
1 vote

Definition of a Katal (unit of enzyme activity)

Although this is old news, as @R.M. has been good enough to provide an answer, I feel I should add my own. The reason is not that I think his answer is incorrect in any way — it is technically quite ...
David's user avatar
  • 26.2k
1 vote

Definition of a Katal (unit of enzyme activity)

A katal is indeed a specific amount of enzyme (rather than a reaction rate), but one which is measured with respect to the catalytic activity of the enzyme, rather than with respect to the number of ...
R.M.'s user avatar
  • 1,554
1 vote

Can you freeze an ATP assay sample for later use?

Pure ATP can be stable when stored frozen but I would be worried, perhaps unjustifiably, about storing a homogenized sample. You should try dividing a homogenized sample in half and assaying one half ...
canadianer's user avatar
  • 17.8k
1 vote

Which analytical techniques could you use to research relationships between 2 proteins?

There are plenty of methods to use. Here is a overview and background of some common methods as written by ThermoFisher: https://www.thermofisher.com/se/en/home/life-science/protein-biology/protein-...
CuriousTree's user avatar
1 vote

How do detergents interfere with protein assays?

Depending on the detergent, its concentration, and the exact assay being performed, it can affect both the protein and the assay reagent(s). Some detergents will bind the (usually colorimetric) ...
MattDMo's user avatar
  • 15.3k
1 vote

Measuring optical density with/without re-suspending cells

Definitely (2). Clumps of cells will cause a lot of noise in your absorbance measurements (depending on whether the light beam hits a clump or some empty space). I don't know what your microbe is or ...
Victor Chubukov's user avatar
1 vote
Accepted

Ways to monitor enzyme kinetics with very fast time resolution?

Some ATPases can work with MANT-ATP or similar fluorescent ATP analogs that change their fluorescence properties upon protein binding as well as hydrolysis of their phosphate group. This has been used ...
Thawn's user avatar
  • 1,239

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