Recent Posts

Pages: 1 2 [3] 4 5 ... 10
21
DiPIA / Re: Fewer concentrations for affinity analysis
« Last post by GE Anna M on October 27, 2015, 01:15:36 AM »
Hi Tamara!
Running steady state affinity is a bit different from running a kinetic experiment. When assessing the kinetics of an interaction you can usually obtain a very good estimate of the rate constants using as little as one concentration, if a suitable concentration with curvature and a long enough dissociation is used. When assessing the affinity of an interaction you need to capture the shape of the curve, i.e. when fitting the different concentrations to the steady state affinity model. You also need to make sure that you are approaching saturation of the surface in order to obtain a reliable value. Thus, a higher number of concentrations is required to assess affinity. The standard recommendation is to use seven concentrations (1,5 x dilution) plus a blank. Deviation from this will result in less reliable results.

/Anna 
22
DiPIA / Forgotten how to startup and prepare a Biacore system for use?
« Last post by GE Marie W on October 19, 2015, 12:07:23 AM »
Maybe forgotten how to start a Biacore system?
Take a look at the new video available on Youtubeit show you how to startup and prepare a Biacore™ T200 system for use.
Biacore YouTube playlist: https://www.youtube.com/playlist?list=PLUSfjij8XMn62-K79jZvK8ur2C9BreHj4
23
DiPIA / Fewer concentrations for affinity analysis
« Last post by Tamara on October 11, 2015, 11:54:28 PM »
Hi!
I am a bit curious about the new Biacore S200 instrument. It seems to have a lot of benefits and interesting features. I have seen some nice examples in which people have used a little a three concentrations for kinetic experiments. This really speed things up! Can I also reduce the number of concentrations for steady state affinity analysis? If so, how low can I go and what should I think about when I choose the concentrations?

/Tamara
24
DiPIA / New Biacore S200 poster
« Last post by GE Anna M on October 11, 2015, 11:43:18 PM »
Have you seen the new Biacore S200 poster "Increased SPR-sensitivity enables fragment screening and kinetic characterization at lower surface densities"? Make sure to download and check it out today! In this poster we describe how we have utilized the increased sensitivity of Biacore S200 to resolve kinetics  for a very low activity target with response levels of less than 1 RU.
We also used the new ABA injection of Biacore S200 to setup a competition experiment for a kinase target with dual-site binders. By blocking the ATP site of the target using the ABA injection, we were able to resolve the kinetics for the compounds binding only to the allosteric site. :)
25
DiPIA / Lead selection of biologics...A new Application note
« Last post by GE Asa on October 07, 2015, 04:59:53 AM »
Hello,

this study was performed at Swedish Orphan Biovitrum, a biotherapeutic company focusing on rare but severe diseases. The application note  describes how Biacore T200 was used in early drug discovery for characterization of biotherapeutic lead candidates. It involves protein receptor specificity, some qualitative kinetics and ranking of protein leads with different N-glycan profiles.

Obtained receptor binding results were correlated with endosomal uptake in fibroblasts as shown using InCell 2000 Analyzer.

Download it from DiPIA Community Home page! (Not this forum "Home", but the very FIRST "Home" :)

Best,
Asa
26
DiPIA / Re: Flow rates for kinetics
« Last post by GE Veronica on October 02, 2015, 06:18:32 AM »
Hi Hayley,

that's right that 30 µl/min has several advantages for kinetic analysis. Compared to a flow rate of 5µl/min it is gives a more rapid switch between buffer and sample, mass transport is improved almost two times and analyte can still be injected for at least 10 minutes.

A higher flow rate, such as 100 l/min, will typically not improve the performance of the assay but it will use more sample and will be restricted to shorter injection times of about three minutes. However, a flow rate of 100µl/min can be beneficial for interactions with very fast offrates (kd>5*10-2) where injection times can be short. Here a higher flow rate may extend analysis and enable determination of somewhat faster off rates.

Best regards,
Veronica
27
DiPIA / Flow rates for kinetics
« Last post by Hayley on October 02, 2015, 01:33:05 AM »
Hi!

I've seen that you recommend 30µl/min for kinetics. Are there situations where I should use another flow rate?

Regards,
Hayley
28
DiPIA / Kinetics - the RI factor and Chi2, any recommendations?
« Last post by GE Asa on September 23, 2015, 07:15:21 AM »
When is my fit good?
 
This is how I, no big fan of equations, usually think around this. There is no general recommendation for Chi2 range. Chi2 is in RU2 and should be judged in relation to the response levels. If all data are within 20 RU and Chi2 is 16 RU2 then Chi2 is significant (square root of 16 is 4 compared to 20 RU = 20 % average deviation in the fit). However a Chi2 of 16 RU2 may be ok for 200 RU response level data (4 compared to 200 RU = 2 % average deviation).

If Chi2 is high look also at the residuals and judge if residuals are evenly distributed or if there are large local deviations perhaps caused by disturbances that could be removed.

I see 4 different scenarios for handling of RI factor:

1)   If the association phase and the dissociation phase are nicely connected at the end of injection when looking at the experimental data (the dissociation is starting immediately at the top of the sensorgram at end of injection without any visible “jump”), then no bulk shifts (RI shifts) exist in the experimental data and the RI factor should be set to zero before fitting (in Parameters, RI set to Constant, 0).

2)   If responses consists of square injections (no visible dissociation) then RI should always be set to 0.

3)   This is the tricky one...If it is hard to judge by looking at the sensorgram whether there is a bulk shift in the experimental data or not then RI default setting is used, “Fit local”. After fitting Chi2 may be low and if also RI values are reasonably low in relation to the response levels then all is usually well and the sun is shining and so are you! But if Chi2 is low and instead RI values are high and significant compared to the response levels (as read in the Report table or visualized in the Tools/Components plot), then the fit is still not good. Look at raw data, are bulk levels very high so that large values are subtracted in the reference subtraction? If so try to reduce bulks, normally 100 -200 RU bulks are subtracted without problems. Also check blank subtractions.

4) If there are visible jumps at beginning and end of injections then RI default setting is also used.

Please add any thoughts you may have on this wonderful world of trying to understand your data  :)

/Asa

29
DiPIA / How to judge my kinetic results - The U value
« Last post by GE Marie W on September 22, 2015, 01:31:46 AM »
How to judge my kinetic results!
There are basically three sets of criteria that you should use in assessing the fitting results:
-   The agreement between the experimental and fitted curves, including the shape of any deviations
-   A set of statistical parameters relating to the quality of the numerical results (these are summarized for simple fitting models in a Quality Control Report in newer Biacore systems)
-   The biological and experimental relevance of the results

Here we take a closer look at the U-value:
What is it? A statistical parameter that gives an estimate of the uniqueness of reported parameters. It is determined only for fitting to the pre-defined 1:1 binding model (is not reported in BIAevaluation).
What does it do? For the 1:1 interaction model, the U-value looks for correlation between the separate rate constants (ka and kd)  and Rmax, and reports a high value if any two of these parameters are correlated. If they are, you will not be able to determine unique values for the parameters.
U-value above ~ 25: indicate that absolute values for two or more of the parameters (rate constants and Rmax) are correlated and cannot be determined.
U-value below ~ 15: parameter values are not significantly correlated so can be uniquely determined.

30
DiPIA / Biacore videos on YouTube
« Last post by GE Marie W on September 15, 2015, 04:13:04 AM »
Did you know that we have several great Biacore videos available on YouTube?
Just click on "Videos" on this page www.gelifesciences.com/bcappsupport, then you can see them all (e.g. Principle of Surface Plasmon resonance (SPR) used in Biacore systems).
Pages: 1 2 [3] 4 5 ... 10