Love pure pie . . . "Loads and samples" of dry goods pastes

may often encounter a question when doing a layering experiment: How many samples can be taken in an experiment? More is not less is not, the sample more will cause a part of the sample loss or not achieve the expected separation effect, the upper sample can not fully use the analysis column but also increase the repeated experiments or the use of larger analysis column, really is a tangled thing. What we're going to introduce to you today is how different layers of analysis should determine the sample amount.The sample amount on the -layer column is determined by the volume of the column and the load of the
filler
used
- column volume (CV): the total product of the filler filled in the column is calculated according to the cross-sectional area of the column and the height of the filler filling.
load: the ability of fillers to bind to the intended molecule under certain conditions, divided into static load and dynamic load.. Static load (effective load):under certain conditions, can be combined with the number of real samples on the laminate filler, it is the use of real samples and media fully immersed saturation, and then determine the total number of samples combined on the filler. Factors such as pH, ion strength, additives, and samples can all affect the effective binding load. The experimental conditions change and the effective binding load changes.dynamic load: the numbersamples that can be combined on the analysis column under certain operating conditions. In addition to pH, ion strength, additives and samples, the operating flow rate also affects the dynamic binding load. In general, the lower the flow rate, the higher the dynamic binding load. If the flow rate is close to zero, the dynamic binding load is equal to the effective load..The stratography is carried out at a certain flow rate, so the dynamic load of the filler can be used more effectively for the determination of the sample amount. For a particular filler or analysis column, the instruction manual will give its dynamic binding load for a particular protein, and give the corresponding measurement conditions, which can be used as a reference for the sample amount in the actual experiment. The following illustration shows the dynamic load and measurement conditions of HisTrap HP:
. If it is necessary to determine the binding load of the filler for a particular sample under certain conditions, the actual dynamic binding load can be determined by: (1) Step 1: Guaranteed timeon the KTA The column of HiTrap connected to the corresponding filler or the column of the small test (if process amplification is involved, the height of the small test column should be consistent with the height of the industrial column, ensuring that the retention time of the sample before and after the enlargement is the same as the contact time between the sample and the filler).(2) Step 2: Fully balance the columnbalance the column with a combined buffer (balance buffer) of 5 column bed volume.. (3) Step 3: Calculate the dynamic loadcontinuous sample (real sample) at a certain flow rate (e.g., linear flow rate of 300 cm/h) and use the collector to collect the penetrating fluid to determine the content or activity of the target protein in the penetrating fluid.
At the beginning of the sample, the 280nm curve (A280) rises, at which point the target protein is mostly bindned to the bar, so the target activity (Activity) is almost impossible to detect, and with the increase of the upper sample volume, the load of the column is gradually reached, the activity of the target protein can be detected in the penetration curve. QB, 5%, i.e. a dynamic binding load under certain buffer conditions at a certain flow rate is obtained, calculated as: C0 - concentration of the target protein in the sample (concentration target of solute in feed).. VA - The total product of the sample when the target sample activity in the penetration curve reaches 5% C0 (volume applieduntil the conc. Of target solute in effluent has reached 5% of which C0).. VC - Total column bed volume. . QB, 5%: QB, 5% or QB, 10% can generally be calculated as a dynamic combined load based on real targets or actual processes. The volume of the column used and the load of the corresponding filler are determined, and the amount of samples in the process of layering is easily determined. . After chatting about the relationship between the sample volume and volume and the filler load on the column, let's take a look at how different types of analysis experiments should ensure the upper sample volume according to the five principles of layering. (1) Non-adsorbent layering - gel
filtration -part separation: for example, desalination, buffer replacement and other experiments, the upper sample volume of up to 30% of the column volume, usually recommended for 10%-20% column volume. . Fine separation: If high-resolution part separation is performed using gel filtration, a sample volume of 0.5%-4% of the volume of the column is recommended, depending on the type of the column and the nature of the sample. Usually in order to obtain a better resolution, the sample volume does not exceed 2% of the total column volume. . Analysis of separation or complex sample separation: 0.5% of the total column volume sample is recommended to achieve better separation results. Sample volumes below 0.5% usually do not increase resolution. . Note: Due to the limited volume of the sample on gel filtration analysis, the sample can usually be concentrated at a concentration below 70 mg/mL, which is usually acceptable, but is determined according to the specific nature of the sample (considering sample stability, sample viscosity, etc.). . (2) Adsorptionic the filler load and column volume used in the process. (3) Ion exchange or hydrophobic layering real sample: For a given ion exchange or hydrophobic filler, the true sample volume is generally 5 to 40% of the effective load (because impurities with the same property charge are also combined on the column). . Sample size: The more complex the sample, the smaller the proportion of the sample. If the sample is simple and the proportion of the target protein is large, the upper sample volume can reach 80% of the load. . Load and sample percentage: Fillers combine 1 ml of columns with a load of 25 mg/ml to sample 20 mg if 80% of the load is used, 10 mg samples can be sampled if 40% load is used, and samples of 1.25 mg can be sampled if 5% of the load is used. . Determining the dynamic binding load of a sample: Or, if the dynamic binding load of a real sample is measured, more than 70% of the dynamic binding load can be used directly to sample. . (4) The maximum sample size affinity and affinity and laminate samples is determined by the combined load of the filler. . For example: Ni Sepharose HP fillers can adsorption 40 mg of label protein per milliliter filler, e.g. with 5 ml HisTrap, the maximum sample size is 200 mg, and if the sample concentration is 5 mg/ml, the maximum sample volume can reach 40 ml. . However, in fact, the sample size varies according to the complexity of the sample. In addition, if you use small columns to purify large volumes of samples, sample preparation is very important, because a large number of impurities can cause column blockage, thereby increasing cleaning costs and reducing service life.
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