Q: I have a question for Dr. P. 1. The protein that I get from Dr. P, is that the total protein? 2. Can I extract DNA/RNA/Protein from mammalian cell using Dr. P? If so, can you send me the protocol.
A: 1. Yes, it's total protein. 2. Yes, it can be used to extract all 3 components from mammalian cell. You should have received a manual with the sample. If not, it's available on our website. According, to my supervisor, Dr. Zhongdong Liu, the manual on our website should provide you with the information you need. There's a section called Recommended Protocol. Here's the link to save you time. http://www.biochain.com/biochain/ProtocolManuals/DrPKitManual.pdf
Q: When the customer measured the concentration of the nucleic acid (DNA or RNA), he obtained various concentrations from the spectrometer. Is it normal to obtain various concentrations? What would cause variances in concentrations?
A: The concentration can vary in certain range. Please ask customer dissolve the RNA or DNA completely in 10 mM Tris buffer, pH 7.5. This will help.
Q: Can the Dr. P kit be used for any animal tissue?
Q: Can the Dr. P kit be used to extract from fresh blood? How?
A: The customer should still follow the user's manual except doesn’t need to homogenize the blood sample because the solution can lysis the sample directly.
Q: How does BioChain ensure that there is no protease or phosphostase presence in the extracted sample.
A: The protein lysate from Dr. P kit is denatured protein and from previous steps for RNA/DNA isolation all the protease are “killed.” The protein lysate is dissolved in high SDS concentration buffer. SDS can inhibit any other protease and therefore there is no need to add any other protease inhibitors in the buffer.
Q: Are the ionic channels present in the protein fraction? The customer needs at least 100 µg of proteins. What's the quantity of cells required?
A: The protein isolated from Dr. P kit is total protein . Theoretically ionic channel protein should be present in the fraction as well. But all the protein isolated from Dr. P kit is denatured and inactive. The protein is only good for western blot analysis but not good for any activity assays.
Q: Is DTT or 2-mercaptoethanolcontained in any buffer?
A: Solution 1 contains 2-mercaptoethanol
Q: What's the minimum quantity of cells or tissue you would need to isolate DNA, RNA and protein with your kit ref. K2021010, Dr. P.?
A: The minimal amount really depends on tissue type. For example, for the high yield tissue liver, 50 mg should be ok, and you can reduce the reagent amount proportionally. But for low yield tissue, such as adipose, I think you may have to use 500 mg.
Q: The customer will work with cells. The manual indicates ….0.5 ml Solution 2 per gram cells …. How many cells would you recommend as starting material (10 to the 6 or 10 to the 7)?
A: 10 to the 7
Q: 1)Regarding the protein extraction, is the extracted protein biased to a some specific protein(e.g. DNA binding protein etc)? The customer would like to refer to the comparison data with usual protein extraction reagent like RIPA. 2)Please let us know
A: 1) No. 2) Yes. Unfortunately we don’t have any citation for this kit, yet. 3) When DNA and RNA extraction, is there a possibility that the protein is lost?
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.
Q: I'd like to detect the level of phospho protein level after extracting the protein lysate. If I use BioChain's Dr. P Kit, will the phospho protein still be maintained?
A: Isolation of Protein Lysate to be used for detection of phospho protein level for later.
Q: Which kit was used to isolate the total RNA?
A: We use Dr.P kit, a patent pending technology, to isolate the RNA. The isolated RNA can be used for mRNA isolation, probe generation, RT-PCR, Northern blot analysis, primer extension, RNA protection assay, and In vitro translation.
Q: Please elaborate on step 12 of the protocol. How can the RNA and DNA concentration be adjusted to 0.6µg/µl as they are measured separately? Should this be ‘at least 0.6 µg/µl’? And what if e.g. DNA concentration is high and RNA concentration low?Or how i
A: At this step, the solution is a mixture of DNA and RNA, but you can still determine the concentration by UV260 using the RNA concentration formula which is 1OD=40ug/ml. The solution 3 is specifically for RNA precipitation, and we optimize the best RNA concentration is around 0.3 ug/ul, since the solution we got from step 12 is a mixture, we can only assume DNA and RNA is half and half, so we set 0.6 ug/ul, which means there are about 0.3ug/ul is RNA while the other 0.3ug/ul should be DNA. So 0.6 ug/ul doesn’t have to be exactly, a little bit more and less should be fine.
Q: What's the buffer?
A: Guarnidine lysis buffer
Q: What's the buffer?
A: Sodium Acetate buffer
Q: What's the buffer?
A: Sodium Chloride buffer
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.