E. coli expresses foreign protein, and when sonicated, it is suspended in PBS containing 1% triton-X-100, and then the effect of ultrasound is better.
Well, the effect of 1% triton-X-100 is still very obvious, and it works for some other bacteria, such as Streptomyces.
The bacterial pellet was directly added to the sample 1buffer, plus 5ul of mercaptoethanol, mixed, centrifuged, boiled for 10min, directly loaded, stained bleaching step
The steps are as follows: put the glue in a microwave oven with an appropriate amount of dyeing solution for 1 min (the next time you need to make up the acetic acid), and replace the staining solution with a large amount of water (tap water)
Yes) It can be cooked in the microwave for 10 minutes.
After the expression of the recombinant protein, the cells were disrupted by ultrasonic wave, using an ice bath, 400w, and breaking for 2s for 1s, but a large amount of foam was generated in a short time, affecting
The crushing power, pbs and tris buffer are the same, and finally the broken is not complete, and my target protein is in these unbroken cells.
1* will generate bubbles because your probe position is not set. The probe must be close to the bottom, about 1cm (I am usually 0.5mm from the bottom). Work
The rate will vary depending on the instrument, but you can observe the liquid level, fluctuating but not too intense.
2* break 3S to stop 10S, break a twenty or thirty times to see.
3* The position of the horn should also be noted. If the sound is wrong, it should be adjusted in time. In addition, it can be considered from the viewpoint of the concentration of bacteria. When broken
Try to increase the volume, the best not to exceed 60%.
4* Try to stop 8s for 8s. For some bacterial proteins, your method is difficult to dissipate heat, causing protein denaturation to produce bubbles. It is best to pause for a little longer.
These conditions are more common in proteins in the form of inclusion bodies.
What are the conditions for the sonication of Streptomyces (actinomycetes)?
What are the conditions for the sonication of Streptomyces (actinomycetes)?
Pretreatment is generally a suspension of bacteria configured to a certain concentration.
The specific conditions used when using ultrasonic disruption are:
(1) Bacterial 24 h culture solution was centrifuged at 5 000 r/min for 5 min to collect the cells.
The specific conditions used when using ultrasonic disruption are:
(1) Bacterial 24 h culture solution was centrifuged at 5 000 r/min for 5 min to collect the cells.
(2) Washing with Na2HPO-NaH2PO buffer at pH 7.5 for 3 times, and then using the buffer to prepare the bacterial suspension of 1:3. Placed at 40
mL large plastic test tube.
(3) Place the large plastic test tube in an ice bath and use ultrasonic to break (power 200 W, 1/2" probe, crush for 30 s, intermittent 30 S).
(4) The crushed liquid was centrifuged at 12 000 r/min for 30 min at high speed to collect cell debris and supernatant.
The ultrasonic sterilization process is basically the same as above, that is, the washing cells can also be washed once with pre-cooled physiological saline or pH 8 Tris-HCl.
can. In addition, the ultrasonic dose varies with the amount of sample and the bacteria, the power can reach 400-600w, exceed 5s, stop for 5s, ice bath, to add end
A concentration of 1 mM PMSF. In order to determine the appropriate intensity and frequency of ultrasound, it is necessary to observe at any time whether the cells are completely broken.
Actinomycetes belong to the (G+C) molar percentage (mol%) of Gram-positive bacteria on the phylogenetic tree of prokaryotic systems
(Eubacteria) branching group, which has the molecular and biological characteristics unique to prokaryotes, but in its different groups, the chemistry of cell walls
The composition varies greatly.
In the case of E. coli ultrasound, the method of 400W, super 5 stop 5, the effect is good, but used on Streptomyces, it seems to have no effect
In the case of E. coli ultrasound, the method of 400W, super 5 stop 5, the effect is good, but used on Streptomyces, it seems to have no effect
fruit. Will it be due to differences in cell wall composition, because E. coli is a Gram-negative bacterium.
Another microscopic examination is to test the crushing effect, but is there a direct relationship between the degree of cell disruption and the enzyme acquisition I need? Long break time will also affect
Another microscopic examination is to test the crushing effect, but is there a direct relationship between the degree of cell disruption and the enzyme acquisition I need? Long break time will also affect
Enzyme activity. So I would like to ask anaisai comrades, you provide the "power 200 W, 1/2" probe, broken 30 s, intermittent 30 S" strip
The piece seems to be used to break the spores of Streptomyces, can it be used to ferment the slime after centrifugation? If you can, the whole time of your broken is probably more
Less?
If you need an intracellular enzyme, there is basically a proportional relationship between the degree of cell disruption and the required enzyme acquisition. The long break time does affect
Enzyme activity. This requires a judgment between the optimal crushing time and the enzyme activity. The most straightforward method is to first draw the relevant curve (enzyme activity and
Time curve).
In the experiment, the coryneform bacteria (also G+ bacteria that are difficult to break) are broken, and the crushing time is controlled at about 30 minutes, and the enzyme activity is good.
In the experiment, the coryneform bacteria (also G+ bacteria that are difficult to break) are broken, and the crushing time is controlled at about 30 minutes, and the enzyme activity is good.
If it is a matrix hyphae, it is better to consider using lysozyme.
Generally speaking, these methods can be read:
Generally speaking, these methods can be read:
First, liquid nitrogen grinding
Second, broken with the french press
Third, ultrasonic fracture
Fourth, lysozyme treatment pretreatment
Ultrasonic waves are an elastic mechanical wave in a matter medium, which is both a form of wave and an form of energy. The effect of ultrasound on cells is mainly
Ultrasonic waves are an elastic mechanical wave in a matter medium, which is both a form of wave and an form of energy. The effect of ultrasound on cells is mainly
There are thermal effects, cavitation effects and mechanical effects. The thermal effect is that when the ultrasound propagates through the medium, the friction forces the molecular vibration caused by the ultrasound.
Part of the energy is converted to local high heat (42-43 ° C). The cavitation effect is the formation of vacuoles in the living body under ultrasound irradiation, with the vibration of the vacuoles and
Its violent explosion produces mechanical shear pressure and turbulence. In addition, when the cavitation bubble bursts, it generates instantaneous high temperature (about 5000 ° C) and high pressure (reachable).
500×104Pa), which can thermally dissociate water vapor to produce .OH radicals and .H atoms, redox reactions caused by .OH radicals and .H atoms.
It can lead to polymer degradation, enzyme inactivation, lipid peroxidation and cell killing. The mechanical effect is the primary effect of ultrasound, and the ultrasound is intervening in the propagation process.
The alternating compression and extension of the mass point constitutes a pressure change that causes damage to the cell structure. The strength of the injury is closely related to the frequency and intensity of the ultrasound.
turn off.
100 g of Escherichia coli was dissolved in 1 L of the disrupted solution (broken mash: 50 mM Tris-Cl (pH 8.5) 5 mM EDTA 0.14 M NaCl), stirred
100 g of Escherichia coli was dissolved in 1 L of the disrupted solution (broken mash: 50 mM Tris-Cl (pH 8.5) 5 mM EDTA 0.14 M NaCl), stirred
Mixing, found that the bacteria liquid is sticky, the bacteria can not be well dispersed, broken with a high-pressure homogenizer, after the pressure, the bacteria liquid can not enter the homogenizer, the speed is very slow,
What is the reason? Can there be a good solution, quickly use a homogenizer to quickly break the bacteria?
The amount of the crushing liquid is increased, and the amount which is not well dispersed may be too large. Ultrasonic treatment for 5-10 minutes, the viscosity of the bacterial liquid can be greatly reduced, and can also be promoted
The cells are dispersed.
The power of different models is different. The size of the power determines the size range of the horn (2mm to tens of millimeters in diameter).
How large the horn is used to adjust the scale of the device to this scale (very simple)! The size of the horn determines the processing capacity of 5ml and chooses 3mm, 5~
50ml optional 6 ~ 8mm, 50ml above select 10mm horn, and so on! The duty cycle does not care, mainly refers to the ultrasonic time and stop
The ratio of time.
Yeast broken problem
In general, PROTOCOL uses glass beads to break up yeast cells sigma G-8772. The yeast has a good crushing effect.
The method is still glass beads, which is highly efficient and does not affect the activity of the target protein. This method should generally be used.
For the chemical cleavage method, 10 g of yeast was added with 1 ml of ethyl acetate, and the mixture was thoroughly stirred to a liquid state. The cracking effect of this method is good.
For the chemical cleavage method, 10 g of yeast was added with 1 ml of ethyl acetate, and the mixture was thoroughly stirred to a liquid state. The cracking effect of this method is good.
Add urea lysate (urea 8mol / L, NaCl 0.5mol / L, Tris 20mmol / L, EDTA 20mmol / L, 2% SDS, PH value
8.0), it is said that the effect can be.
Pichia pastoris cell disruption
The problem encountered in the crushing is that the bacterial concentration is too low, and the OD620nm is between 4-6. The minimum crushing volume of the cell disrupter is about 4ml, which
The sample is diluted twice, and the OD is 2-3. We suspect that the broken protein and enzyme activity will not be measured. So ask everyone when the cell is broken.
What is the minimum concentration of bacteria? Our bacteria is a corynebacterium. Once broken, you can concentrate the liquid in a dialysis bag.
The method we did was to dissolve the centrifuged cells (e.coli) in an ultrasonic buffer (50 mM phosphoric acid pH) at a ratio of 1:20.
8.0) 300w 10s/10s broken for 20 minutes. Do a microscopic examination! Basically all broken! I am doing protein purification, making inclusion bodies. In experiment
Our bacterial cell concentration is relatively independent, and has nothing to do with the bacterial od in the culture solution, and does not accept the restriction, which is convenient for the operator to regulate.
Generally, 5-10 ml of lysis buffer is added per g of wet cells.
Ultrasound will definitely produce heat, so there must be a cooling device unless you need the ingredients to withstand high temperatures.
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