Frequently Asked Questions
Mass Spectrometry (MS)
Tandem mass tag technology is used to individually label different protein samples so they can be combined into a single sample for LC-MS analysis allowing for higher sample throughput, less instrument analysis time, high precision of peptide quantitation among replicates, and fewer missing quantified proteins among different samples.
Relative quantitation compares the amount of an analyte in different samples. Absolute quantitation measures the actual amount of an analyte in a sample using a known standard at different concentrations. Label-free, SILAC and TMT workflows are used for relative quantitation of protein samples. Heavy (i.e. AQUA) peptides are used as standard for absolute quantitation of target proteins.
TMT and TMTpro reagents are different chemicals with different masses, therefore it is not recommended to mix the two tags as the combined samples are significantly more complex resulting in fewer quantified proteins.
Label-free, TMT reagents, and TMTpro reagents can be used with any protein sample type. SILAC and Neucode can only be used with cell lines & model organisms that can be metabolically labeled.
SILAC can be used to multiplex 2 to 3 samples. TMT reagents can be used to combine 6 to 11 samples. TMTpro reagents can be used to measure up to 18 samples simultaneously.
This is most often done through use of a “pool channel” for each multiplex set. This reference channel typically contains an equimolar mixture of all samples which is labeled with one of the TMT tags that is shared among the sets. The pool channel can then be used to normalize relative protein abundance measurements across multiplexed sets.
Yes, please use the protease inhibitors in the buffer(s) for preparing protein lysate to block protein degradation.
The protease inhibitors added in your sample buffers will not affect the sample process on our end.
Both methods provide robust results for immunoprecipitation. Biotin tagged antibodies with streptavidin beads provide lower background but require more upfront sample prep, while protein A/G beads are robust and straightforward in use but result in higher background.
Digestion efficiency is optimized through the proper amount of added enzyme and digestion times, maintaining the correct pH for digestion, and avoiding protease inhibitors and strong denaturants.
Proteome Discoverer
Proteome Discoverer
Placeholder
No, we will reduce samples with the appropriate reagent and ensure alkylation according to our standardized procedures.
Trypsin or Trypsin/LysC mix are most commonly used for proteomic applications in order to assure reproducibility and complete digestion. Other commonly used enzymes include Chymotrypsin, Pepsin, LysN, AspN, and GluC.
Yes, MS analysis can be used for evaluation of depletion performance. Protein yields after depletion level can be measured using the BCA assay.
There are many strategies to extract Membrane proteins (MPs) from eukaryotic cell lines and tissues, including subcellular fractionation (e.g., sucrose or sorbitol density ultracentrifugation), cationic colloidal silica absorption, and aqueous-polymer two-phase system. Commercially available kits can be also used for membrane protein extraction.
Mass Spectrometry (MS)
Tandem mass tag technology is used to individually label different protein samples so they can be combined into a single sample for LC-MS analysis allowing for higher sample throughput, less instrument analysis time, high precision of peptide quantitation among replicates, and fewer missing quantified proteins among different samples.
Relative quantitation compares the amount of an analyte in different samples. Absolute quantitation measures the actual amount of an analyte in a sample using a known standard at different concentrations. Label-free, SILAC and TMT workflows are used for relative quantitation of protein samples. Heavy (i.e. AQUA) peptides are used as standard for absolute quantitation of target proteins.
TMT and TMTpro reagents are different chemicals with different masses, therefore it is not recommended to mix the two tags as the combined samples are significantly more complex resulting in fewer quantified proteins.
Label-free, TMT reagents, and TMTpro reagents can be used with any protein sample type. SILAC and Neucode can only be used with cell lines & model organisms that can be metabolically labeled.
SILAC can be used to multiplex 2 to 3 samples. TMT reagents can be used to combine 6 to 11 samples. TMTpro reagents can be used to measure up to 18 samples simultaneously.
This is most often done through use of a “pool channel” for each multiplex set. This reference channel typically contains an equimolar mixture of all samples which is labeled with one of the TMT tags that is shared among the sets. The pool channel can then be used to normalize relative protein abundance measurements across multiplexed sets.
Yes, please use the protease inhibitors in the buffer(s) for preparing protein lysate to block protein degradation.
The protease inhibitors added in your sample buffers will not affect the sample process on our end.
Both methods provide robust results for immunoprecipitation. Biotin tagged antibodies with streptavidin beads provide lower background but require more upfront sample prep, while protein A/G beads are robust and straightforward in use but result in higher background.
Digestion efficiency is optimized through the proper amount of added enzyme and digestion times, maintaining the correct pH for digestion, and avoiding protease inhibitors and strong denaturants.
Proteome Discoverer
Proteome Discoverer
Placeholder
No, we will reduce samples with the appropriate reagent and ensure alkylation according to our standardized procedures.
Trypsin or Trypsin/LysC mix are most commonly used for proteomic applications in order to assure reproducibility and complete digestion. Other commonly used enzymes include Chymotrypsin, Pepsin, LysN, AspN, and GluC.
Yes, MS analysis can be used for evaluation of depletion performance. Protein yields after depletion level can be measured using the BCA assay.
There are many strategies to extract Membrane proteins (MPs) from eukaryotic cell lines and tissues, including subcellular fractionation (e.g., sucrose or sorbitol density ultracentrifugation), cationic colloidal silica absorption, and aqueous-polymer two-phase system. Commercially available kits can be also used for membrane protein extraction.
DNA Sequencing
Our partner, Quintarabio, houses customer accounts on their website. Click here to register for a new account. (https://www.quintarabio.com/user/register)
Create an account (if needed) and log in to complete the online order form or import the order information from an Excel file. Prepare your sample according to the Sample Preparation Guidelines. Take advantage of our local pick-up options, or ship to 5350 Partners Court, Suite C
Frederick, MD 21703.
Poochon Scientific is continuously striving to provide the highest quality DNA sequencing service, fastest turnaround times, and overall value for our customers. Our experienced staff and state-of-the-art equipment run 24/7 to provide you with the superior results you deserve and optimize the most complex reactions. Other companies may offer lower prices for similar sequencing, but they can only do so by cutting corners, using cheap reagents, and offering no Quality Control. All too often mistakes on their part will leave you with the wrong data or have you wasting time solving their issues by yourself.
We offer 5 free reactions to all new customers that want to try our DNA sequencing service. Just set up an online account, submit an order, and experience the quality Poochon Scientific provides.
We use ABI 3730xl DNA Analyzers, the Gold Standard for high throughput genetic analysis.
Nanopore Sequencing
PlasmidExpress – this service is for sequencing analysis of full-length plasmids, up to 30kb, by Nanopore long-read sequencing technology, the 3rd Generation Sequencing Technology.
htps://www.poochonscientific.com/services/dna-sequencing/nanopore-sequencing/. The link will direct you to the resources you need regarding the PlasmidExpress service. Click “Place an order” to place your order online. Poochon Scientific and Quintarabio partner to offer this service. Please use Quintarabio’s website to place orders and check results. Poochon is responsible for sample pick-up, analysis, billing, and customer service.
Please use the Poochon-Quintara sample drop boxes (link to the dropbox location list). Pickup times are the same as Sanger sequencing. The cut off time for NIH is 5:00 PM and the cut off time for JHU is 4:00 PM. Please email [email protected] if you have any questions.
The turnaround time is 24 hours for standard plasmid samples.
Nanopore long-read sequencing technology is used to provide PlasmidExpress, whole plasmid sequencing service. No primers are required. Using Nanopore technology, it is possible to sequence through ITRs, LTRs, large repeats, and hairpins. However, there may be difficulties sequencing homopolymer regions such as a Poly-T (TTTTTTTTTTT) region, for which we recommend confirming potential artifacts by Sanger sequencing.
OD260/280 = 1.80 – 1.90; Concentration: 30-100 ng/µl, 10 µl • For custom service sequencing of very large circular plasmids, ≥100 ng (30-150 kb) /µl, 10 µl, or ≥200 ng (150-300 kb) /µl, 10 µl.
• For double-stranded DNA, normalize to the specific concentrations listed above.
• Please refer to published literature for plasmid extraction protocols. Submit the final purified plasmids in elution buffer (10 mM Tris, pH 8.5) or nuclease-free water; avoid buffers containing EDTA (e.g. TE or AE buffer) whenever possible.
• Plasmid samples are sequenced WITHOUT primers, so please DO NOT ship any primers with your samples or mixed into your samples.
• Our low sequencing prices and fast turnaround times do not include DNA extraction or quality control (QC) services, so please verify with full QC that your samples meet the following requirements prior to shipping.
• For best results, samples should NOT contain any of the following:
• RNA (RNase treatment is recommended during extraction)
• Denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.)
• Residual contaminants from the organism (heme, humic acid, polyphenols, etc.)
• Insoluble material, colors, or cloudiness
• Samples should also be “pure” in the sense that they should only contain copies of a single clonal plasmid molecule.
• Sending mixtures of molecular species will give mixed results and is at your own risk!
Please click here for details on reading results.
Data is automatically emailed once the sequencing is complete and run through our rigorous QC procedure. You can directly download your data in a secure atachment, and you will receive a custom sequence summary from a member of our QC team.
The price per sample is $15. You may use the same standing PO number with us. We accept credit cards, standing purchase orders, and checks for payment. Feel free to contact us for a quote, if needed.
Please use the NCBI BLAST tool to blast the target sequence against the PlasmidExpress resulted sequence.
Please email us at [email protected] and provide the order ID and sample name as well as the predicted missed sequence (gap) and predicted full-length sequence of the plasmid tested. Our technical team will answer your inquiry within 24 hours.
(1) Scientific rigor and peace of mind.
(2) Identify any possible mutation sites outside of the gene analyzed by Sanger sequencing which could be occurred during cloning.
(3) For target DNA insertion longer than 1 kb, instead of multiple Sanger runs or synthesizing a sequencing primer or doing primer walking, sequence the whole plasmid not only save time but also save cost.
Our service is intended for purified plasmids prepared from a clonal population of molecules. You can send mixtures of molecular species, but we can’t predict the analysis outcome, so it’s at your own risk. There two most common outcomes:
1) If your species are very similar (e.g. differ by only a few nucleotides), the pipeline will most likely create a single SampleName_contig.fas file (one contig) with low confidence positions at SNP/in/del locations. You can view those locations in your provided sampleName_contig_chrom.csv and sampleName_contig_lowc.csv.
2) If your species are sufficiently distinct (e.g. vastly different in size or sequence), the pipeline will most likely create more than one contigs, SampleName_contig001.fas, SampleName_contig002.fas etc. Ultimately, which species end up producing a consensus will vary depending on overall sample quality, coverage, and relative abundance/degradation of each species.
3) Sequencing is considered successful if the pipeline is able to generate any consensus contigs, even if it is not your target. Re-sequencing mixtures won’t change the relative proportions of the species, but you can submit multiple aliquots if you need higher total coverage. If the pipeline does not produce a consensus for your target, you can analyze the raw reads from your provided SampleName_reads.fastq.gz by your own tools.
The most common error modes for Oxford Nanopore are deletions in homopolymer stretches and errors at the Dam methylation site GATC. This limitation is expected to improve with future updates to their sequencing chemistry and basecalling software.
We do not guarantee any specific level of coverage. The number of raw reads generated can vary substantially depending on sample quality. Successful samples sent at the required concentration typically yield in the high dozens to hundreds (or thousands!) of raw sequencing reads. Final coverage of the consensus depends on how many of the raw sequencing reads are full-length plasmids and whether any degraded plasmid reads can be aligned to the full-length consensus.
: Six data files will be generated for each plasmid sample.
1) A Summary as “.pdf” – A report with the summary of results and data
2) One or more than one contigs as “contig.fas” – A continuous sequence with annotations resulting from the reassembly of the passed reads generated by the Nanopore sequencing analysis [Note: You may use SnapGene viewer (free download: https://www.snapgene.com/snapgene-viewer), or other software to visualize the annotation as a plasmid circle map or linear map to analyze the sequence]
3) One excel file as “contig._chrom.csv” – List of the whole sequence determined (open via Microsoft Excel)
4) Another excel file as “contig_low.csv” –List of nucleotides with low score/confidence (open via Microsoft Excel)
5) “Reads.fastq.gz” – fastq raw data file
6) “Sample_status.txt” – Data statistics and barcode number.
For plasmids, “failure” refers to the failure of your sample to produce a consensus sequence with at least 10x coverage. Our low sequencing prices and fast turnaround times do not include extensive QC to determine why plasmid samples fail. Although we do not provide definitive reasons for failure, by far the most common reasons are:
1) Samples are not prepared to meet with the required DNA concentration and quality, concentration ≥ 30 ng/µl, OD260/280 = 1.80 -1.90.
2) Samples contain a mixture of plasmid species and/or fragmented genomic DNA or fragmented plasmids. You may see evidence of this failure mode in a wide range of read lengths reported in the raw read length histogram.
It is relatively rare that we cannot return a plasmid sequence, but some rate of failure is unavoidable. We may atempt to re-sequence failed samples if your sample quality and quantity permits (with follow-up results delivered in 2-3 business days). If the sample fails a second time, we will conclude that something about the sample makes it un-sequenceable. Unfortunately, we must still charge for failed samples, since we spend more time and resources on them than we do on successes
