Frequently Asked Questions - Answers to Reagent Product Issues-He Yuan Li Ji

Q：What are the advantages and disadvantages of BCA protein quantification method?

advantage:
1. This method is widely used, easy to operate, fast, and can complete the test within 45 minutes. 4 times faster and even better than the classic Lowery method;
2. Not easily affected by general concentrations of detergents, urea, etc., with strong anti-interference ability;
3. The coefficient of variation for detecting different protein molecules is also much lower than that of the Coomassie Brilliant Blue method;
4. Accurate, high sensitivity, and good reagent stability. The BCA method has a good linear relationship in the concentration range of 20-20 ug/mL, and the range of trace BCA determination is 0.5-10 ug/ml.
5. Economical and practical, in addition to test tubes, measurements can be performed in microplate wells, greatly saving sample and reagent usage;
Disadvantages:
1. May be affected by chelating agents (EDTA, EGTA) and high concentrations of reducing agents (DTT, β - mercaptoethanol);
2. Incubate the reaction in a constant temperature water bath for 30 minutes or longer.
Q：Can mycoplasma clearing agents be used together with bispecific antibodies

Do not mix with antibiotics other than streptomycin bispecific.
Li Ji Bio Quick Cell Mycoplasma Eliminator (1000X) effectively inhibits the replication of Mycoplasma DNA by suppressing the activity of Mycoplasma DNA gyrase. Starting from genetic material, it thoroughly kills Mycoplasma, allowing cells to completely eliminate the troubles of Mycoplasma contamination and ensuring the reliability of test results.
Mainly used to remove mycoplasma from cells, serum, and culture medium, the entire process only takes 3-5 days; This product can eliminate most types of mycoplasma and is non-toxic to the cells themselves.
Li Ji has validated over a hundred types of cells, such as mouse embryonic stem cells or iPS cells, human embryonic stem cells or iPS cells, HEK293, Hela, HepG2, HCT116, COS-7, Vero, Huh-7, MDCK, PANC-1, SW620, U2OS, MCF-7, MRC-5, NIH-3T3, CCC-ESF, CHO-S, CHO-K1, CHO-DG44, H295R, HL60, K562, MDA-MB-231, SP20, T47D, BM, and BV2.
Q：Which reagent kit is better for long-term preservation of tumor tissue activity?

The live tissue cryopreservation kit vitrifies frozen tissues, effectively maintaining their morphological structure and function. The tissue activity can be efficiently preserved from the molecular level to the tissue level, achieving long-term and efficient preservation of tissue activity. It is suitable for cryopreservation of animal and tumor tissues. The live tissue resuscitation kit is used in conjunction with the cryopreservation kit for immediate resuscitation and efficient preservation of tissue activity.
Product features:
Vitrification freezing technology, no need for programmed cooling;
After recovery, the tissue activity can be preserved by more than 80%, which can be used to construct PDX/PDC models, drug sensitivity tests, and single-cell sequencing;
No bacteria, no fungi, no mycoplasma;
Sealed storage at 2-8 ℃, with a shelf life of 1 year;
Q：What are the advantages of the live tissue cryopreservation and resuscitation kit?

Freezing storage:
✔ The tumorigenic ability of PDX remains basically unchanged before and after cryopreservation;
✔ After freezing, it can be stored for a long time;
✔ Can replace frozen cut and paraffin embedded tissues;
✔ Realize the preservation of DNA, RNA, and protein activity;
Recovery:
✔ After recovery, the morphology and function are consistent with fresh tissue;
✔ Primary cells can still be separated after recovery;
Q：Does Li Ji Biology's lentivirus preservation solution contain DMSO?

The buffer components of Li Ji Biology's EZ lentivirus cryopreservation solution have been optimized, using Hepes and sodium bicarbonate for buffering, and adding components such as L-glutamine, trace elements, and cryoprotectants. It does not contain DMSO and is suitable for lentivirus resuspension after ultracentrifugation, PEG precipitation, and ultrafiltration concentration. The slow virus particles resuspended with this product have low toxicity, long storage time, and multiple repeated freeze-thaw cycles.
Q：Does the pre dyed protein marker strip fade on the film during the transfer printing process?

Fading is most likely caused by the detergent in the sealing/washing solution washing some proteins off the membrane. Dyes themselves do not wash off from proteins because they are covalently bound. It has been found that membranes with smaller pore sizes can better retain proteins during closure and washing processes. Therefore, in order to achieve excellent resolution and protein retention, it is recommended to use 0.2 µ m instead of 0.45 µ m membranes. After transfer printing, a pencil can be used to circle the pre dyed strip on the film, so that the position of the strip can still be identified after sealing and processing.
Q：Why is the pre stained protein marker band also exposed during WB experiments?

Pre stained protein markers are composed of recombinant proteins, and the protein markers used may contain His tags. If the primary antibody uses his tag, the secondary antibody will not only bind to the primary antibody with his tag, but also to the protein marker band with his tag. Therefore, after adding ECL luminescent solution, the protein marker and the target protein in the sample will be exposed together.
Q：Can pre stained protein markers be stained with Coomassie Brilliant Blue?

Some pre stained markers can be stained with Coomassie Brilliant Blue, while others cannot. Reason: Coomassie Brilliant Blue contains acidic functional groups, such as two - HSO3 in R-250 molecules. The acidic groups can bind to the basic functional groups of proteins, causing the protein to be colored. The higher the protein content, the darker the color. Pre dyed protein markers are covalently bound to proteins of different sizes and dyes. Depending on the dye, the binding position of the protein is also different. If the dye on the marker binds to the Coomassie Brilliant Blue binding group, it cannot bind to Coomassie Brilliant Blue again for staining. Otherwise, Coomassie Brilliant Blue can bind and stain.
Q：What are the precautions for using pre stained protein markers

1) Due to the coupling effect between pre dyed protein markers and dyes, the migration rate of markers varies in different buffer solutions. Therefore, it is important to choose an appropriate buffer system before use. If the electrophoresis buffer is not mentioned in the user manual, it is recommended to use a non pre stained protein marker to calibrate the molecular weight of the pre stained protein marker first;
2) In low concentration gels, low molecular weight proteins will swim at the front edge of the dye;
3) When performing Western blot on high molecular weight proteins, it is necessary to extend the transfer time or increase the transfer voltage.
Q：How to determine bacterial contamination in cultured cells

After overnight cultivation of cells contaminated with bacteria, the culture medium will significantly turn yellow, and bacterial sediment can be seen with the naked eye in the culture bottle. Shaking the culture medium will cause turbidity, and under a microscope, cells can be observed with fine sand covering the entire bottom of the culture bottle. The distinction between cell debris and contamination can be determined by overnight cultivation. The main components that do not become cloudy overnight are cell debris and metabolites, while those that become significantly yellow and cloudy overnight can be identified as contamination. Bacterial contamination of cells usually erupts to a visible state within 1-2 days, and bacterial contamination generally does not cross contaminate. Timely disposal of contaminated cells is sufficient.

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