Endothelial Progenitor Cell
Total RNA
Total Protein
Northern Blot
cDNA
Compartment Protein Isolation Kit
Dr.P Sets
Frozen Tissue Section
Protein Lysate
Total Protein Array
Paraffin Tissue Section
Paraffin Tissue Array
Western Blot
Other Kits and Reagents
Others

Endothelial Progenitor Cell

Q: How pure is your EPC?

A: Our ICC analysis shows that over 95% of our EPC are positive for EPC markers.

Q: Are your EPC from single donors or from mixed donors?

A: Currently, we provide EPC from individual donors.  We will pool EPC from different donors in the future.

Q: Do we need an immediate wash when thawing cryopreserved EPC?

A: You should not perform the immediate wash when thawing cryo-preserved cells.  However, you should wash once after cells are attached.

Q: We see some cell debris in the culture.  What should we do?

A: Wash with 1 x PBS/1% BSA three times; and change medium daily until the debris disappear.

Q: Are your EPC positive for CD34?

A: Yes

Q: Are your EPC positive for CD133?

A: No. CD133 is a hematopoietic stem cell marker, not specific for EPC.

Q: How do cryo-preserved EPC compare to proliferating EPC?

A: We have done side by side comparison between cryopreserved EPC and proliferating EPC.  There is no difference between them regarding the proliferation rate (doubling time), number of passages, vWF immunostaining, qRT-PCR profile as well as LDL-uptake/lectin binding.  

Q: How many passages can this EPC cell be available?

A: Our EPC can be cultured for up to 6 or more passages after our customers receive them, but we do not recommend using these cells for important experiments after 3 passages.  Decreased growth rate, decreased cell density, and increased cell debris may be observed in late passages, although cell morphology is still good.

Q: How long can you guarantee the quality of frozen cells kept in liquid nitrogen?

A: EPC kept in liquid nitrogen for 3 years do not significantly lose their biological activity.

Q: How about the lot-to-lot difference of this product?

A: Donor variation is very common.  There may be minor lot-to-lot differences, because we develop this product from different donors. The differences include cell proliferation potential (i.e., some lots may proliferate for more passages than others), and cell density.  Different lots derived from the same donor are usually quite consistent. We will not sell those lots with significant differences in their morphology, proliferation rate, or other characteristics.

Q: Will you have any limitations to disclose donor profiles to the customer?

A: What we can provide is the gender, age, and racial information of the donor, and that the donor is negative for HIV, HBV, and HCV. There is no charge for providing this information if the customer requests.

Q: Shall I use your EPC growth medium for maintaining EPC?

A: We designed an EPC growth medium with our supplements, and have tested it for cell proliferation, morphology, and LDL uptake/lectin binding.  To our best knowledge, there is no other commercially available medium specifically designed for EPC growth. We strongly recommend our EPC growth medium for maintaining EPC.

Total RNA

Q: What is the concentration of your Total RNA samples?
A: The Concentration of Total RNAs varies in a range of 1-3 ug/ul.

Q: Can I have the same total RNA from different individuals?
A: Yes.  Usually we should be able to provide total RNAs from major organs from at least 10 different donors.

Q: Can I have the same total RNA pooled from several donors?
A: Yes.  We have some standard total RNA from 5 different donor pool.  And we can also custom make pooled total RNA.

Q: What method did you use for total RNA isolation?
A: The method is proprietary modified phenol extraction.

Q: Is your RNA treated by DNase I?
A: Yes, most of RNAs, especially from the major organs are DNase I treated. However, we don't treat RNAs from rare tissues and some of tumor tissues. 

Q: I used your total RNA to do RT-PCR, but I did not get the PCR product?
A: It is possible that the gene of your interest is a very low abundant gene, increase Total RNA amount in your RT reaction or using our mRNA instead may help.

Q: I got your total RNA, and I measured the DEPC water diluted total RNA concentration again by myself, but the concentration I got is lower than yours, can you tell me why?
A: I would suggest you use our method to measure the concentration:
Dilute the RNA in 10 mM Tris-Cl, pH 7.5 but not DEPC water. We usually took 2 ul RNA into 498 ul 10 mM Tris-Cl, (pH 7.5), then measure the OD, use the OD value x 40ug/ml, and then x250 for the final concentration. Using DEPC water will always result lower UV260/280 ratio and lower concentration, and the concentration measured by DEPC water is not stable.

Total Protein

Q: What are the components of the total protein lysate buffer? And what is the concentration?
A: The protein lysate buffer includes Hepes, MgCl2, KCl, EDTA, Glycerol, sodium deoxycholate, sucrose, NP-40, and cocktail of protease inhibitors. However the concentrations of the components are proprietary.

Q: Is your total protein ready-to-use?  Can I load the protein directly on SDS-PAGE gel?
A: No.  The protein need to be mixed with protein loading dye and heat at 95ºC for 5 minutes, then transferred on ice before loading on the gel.

Northern Blot

Q: How many times can your northern blots be stripped?
A: The house keeping gene can still be detected after stripping the blot 10 times, but we can't guarantee customers can detect the gene of their interest after 10 times stripping. Usually the blot can be reused at least 5 times if over stripping does not happen each time.

Q: Do you have specific hybridization buffer for your northern blots?
A: Yes, we do have FastHyb hybridization solution (Cat# L1031250) which is perfect for hybridizing our northern blot.  However, BioChain's northern blots are almost compatible with any kinds of hybridization buffer, though the background might be increased in some cases.

Q: What sizes of mRNAs on your northern blots?
A: The size mRNAs on our northern blots can reach over 10 kb.

cDNA

Q: What is the concentration of your cDNA?
A: Our cDNA is PCR ready cDNA, 1 ul is good for 1 PCR reaction, since 11 ug Total RNA was used to produce 40 ul cDNAs, the concentration of the cDNA should be about 2.5 ng/ul. 

Q: I used your cDNA as template to amplify my target gene, but I can not get the signal, while I did get good signal of a house keep gene, what is the problem?
A: It is possible that the gene of your interest is a GC rich gene, optimizing your PCR condition by adding 0.5 M Betaine may help you solve the problem.  Also, your target gene may be a low abundant gene, in such case, we would recommend you purchase mRNA either from BioChain or other sources, and use mRNA as template for cDNA synthesis.

Q: If I use the same amount of cDNA for different volume of PCR reaction, will I get different amount or concentration of PCR products?
A: Theoretically, the PCR product amount should be the same amount but the concentration should vary depend on the PCR volume. If a very faint band from the first round PCR was obtained, then a sharper band should be obtained with a second round PCR using the first round PCR product as templates. 

Compartment Protein Isolation Kit

Q: What is the difference between CNM and CNMCS kit?
A: These two kits are exactly the same except CNMCS kit has one more buffer, which is CS buffer for isolation of cytoskeleton proteins.

Q: I am studying mitochondrial proteins, and I would like to know by using your CNM kit, which fraction contains mitochondrial proteins.
A: It is membrane fraction.

Q: What is the components of each buffer in your CNM and CNMCS kit?
A: The information can be obtained from the kit manual.

Q: I would like to know whether the different extractions resulting from CNM kits are adequate for analysis by immuno precipitation? Are the complexes of macro molecules kept for this analysis?
A: Yes, all the proteins extracted by the kit should be ok for i.p analysis. The complexes of macro molecules should be kept.

Q: Do you know if the membrane fraction contains every membrane proteins when you use your CNMCS or CNM kits?
A: Yes it does.

Q: Are all proteins obtained by this CNMCS kit in native conditions or in denatured forms?
A: Cytoplasmic, nuclear, and membrane proteins are in native conditions, but cytoskeleton protein is in denatured form.

Q: Do you have any idea in which fraction the microsome proteins is when you use your CNM or CNMCS kits?
A: We believe the microsome proteins should be in the cytoplasmic protein fraction since the centrifugation speed is not high enough to precipitate the microsome during that preparation.

Q: Can your CNM kit separate the membrane proteins from different organells?
A: It is very hard for us to separate the membrane proteins from different organells, at current situation we don't have any answers for that.

Q: I used your CNM kit isolating compartmental protein, and the protein concentration was measured by UV spectrum. The quantity of the membrane protein was 10 times more than the cytoplasmic protein, is this normal?
A: No, it is not normal. Usually the relative yield of Cytoplasmic and membrane fractions, it's about 2-3:1. I would suggest you run your protein on SDS-PAGE gel and see if the protein intensity matched the concentration you have measured from UV spectrum. If the protein intensity from SDS-PAGE gel is consistent with the quantity measured from UV spectrum, then the tissues or the cells probably were not homogenized very well in the first step. Increasing the speed setting of the homogenizer for tissues, and increasing the repetition of homogenization up to 5 times (normally 3 times). will help. For cells, I suggest pipette enough times to make sure the cells are completely lysed.

Q: Are the proteins isolated by CNM kit good for 2D?
A: Yes, but nuclear fractions need to be dialyzed before using it for 2D experiment.

Dr. P Set

Q: What is Dr. P set product?
A: Dr. P set include 50 ug RNA, 10 ug genomic DNA, and 100 ug total protein. All these 3 items are isolated from the same piece of biomaterial simultaneously.

Q: What is the difference between Dr. P genomic DNA and general genomic DNA?
A: General genomic DNA was isolated from tissues or cells only while no RNA and protein were isolated at the same time. The quality of general genomic DNA is the best. Dr. P genomic DNA was isolated while RNA and protein were also isolated. There are some difference in the isolation methods. 

Frozen Tissue Section

Q: I saw your frozen section was fixed by aceton. Can I get frozen section without fixation?
A: Yes, you can if you mention this request specifically. 

Protein Lysate

Q: What method did you use for total protein and compartmental proteins isolation?
A: We used Total protein extraction kit (Cat# K3011010) for total protein isolation, and CNM (K3012010) or CNMCS kit (K3013010) for compartmental protein isolation.  Compartmental protein isolation is a patent technology.

Q: How did you measure your protein concentration?
A: We Use Bio-Rad DC protein determination kit to measure the concentration of our protein.

Q: What QC method did you use to prove that your compartmental proteins were well separated?
A: A western analysis with GAPDH (a cytoplasmic protein) antibody shows very strong signal in cytoplasmic protein, but very low signal in nuclear and membrane protein. Histon is shown in nuclear, EGFR is only shown in the membrane protein, and vimentin is only shown in cytoskeleton protein, very small amount in membrane protein, but not in cytoplasmic and nuclear proteins.

Q: I need to use trypsin to treat the protein before doing my experiment, since you have protease inhibitor cocktail, I wonder if those inhibitors will inhibit the activity of trypsin?
A: Yes, you need to get rid of the protease inhibitors before doing your experiment in such case.

Q: Can I have one kind of protein from different donors?
A: Yes, we can custom make one kind of protein from multiple donors.

Total Protein Array

Q: What is your total protein array like? Does each spot on the array contain each single protein?
A: The total protein arrays are modified nitrocellulose membranes spotted with proteins from different tissues. Each spot on the array contains a protein pool from an organ, but not a single protein.

Q: How much protein lysate is on each spot?
A: About 16 ng.

Paraffin Tissue Section

Q: How did you treat the tissue to make paraffin tissue section?
A: The tissue was fixed in formalin immediately after harvest, then dehydrate and embedded in paraffin.

Q: What have the sections been coated with? for example, do you use 3-aminopropyltriethoxysilane? 
A: The sections have not been coated with any thing.

Q: how long were tissue fixed after harvest from the body, either through biopsy or autopsy? 
A: At least 48 hours.

Q: What did you use to fix the tissue?
A: Formalin.

Q: Do cerebellum tissue sections contain the following structures: external granular layer, internal granular layer, molecular layer, purkinje layer and deep cerebellar nuclei? 
A: Yes, they do.

Paraffin Tissue Array

Q: How many spots are on each of you tissue array? What is the size of each spot?
A: There are 60 to 96 spots in our tissue arrays, generally most of tissue arrays are 66 spot format. The spot size is 1.5 mm diameter.

Q: Was the heart which is used to make tissue section perfused?
A: No, it was not.

Western Blot

Q: Are the proteins of each lane on the blot from the same donor or pooled donors?
A: Generally, protein of each lane on the blot is from a single donor. However, the protein from rare tissues might be pooled.

Q: How much protein did you load on each lane
A: 50 ug.

Other Kits and Reagents

Q: I saw your manual of Attoglow Western Enhancing Kit that the enhancer works on PVDF membrane, but does the enhancer work for nitrocellulous membrane?
A: Yes.

Q: My membrane was dried after the protein transfer. Will the enhancer still work on my dried membrane?
A: Yes, it does. However, we would recommend you extend the enhancer incubation time from 2 minutes to 5-10 minutes.

Q: What homology does your Human Beta Actin cDNA probe have with rat? 
A: The human beta actin cDNA probe we have share 89% homology with rat.

Others

Q: Are your samples from surgery or death donors? Do you provide information about sex, age and about the surgery/post-mortem time elapsed before processing the tissue? 
A: The normal tissues are from death donors. We do provide sex and age information. The post-mortem is about 4-6 hours. The tumor tissues are from surgery, and they are snap frozen with liquid nitrogen right after disected from the body.

Q: How many matched pairs do you have for each tumor type?
A: We usually have more than 10 different donors.

Q: What is species of your monkey products?
A: We have Rhesus (Macaca Mulatta) and Cynomolgus monkey. There is suffix "cy" in the end of catalog numbers of Cynomolgus monkey products.

Q: What is the species of your mouse products?
A: Balb/C.

Q: What is the species of your rat products?
A: Sprague Dawley, Wistar

Q: What is the species of your dog products?
A: Beagle.

Q: Which part of tissue do you isolate RNA from plants?
A: Leaves.