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.
It is not recommended to add protease inhibitors as they can affect trypsin and trypsin/Lys-C activity.
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.
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.
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.
Our general pricing for our DNA sequencing services can be found on the DNA sequencing service page. We offer many academic and regional discounts, so please contact our sales team for a more accurate quote based on your specific order.
We provide two tiers of QC, first our proprietary analytical software will check for all basic parameters of the reactions, and then the QC manager will review each reaction and troubleshoot those that are not optimal, providing you with a personalized summary of your order. We will work closely with you to optimize any reaction that you need. Visit our Data Sample page to see the value in our service.
Once you place an order online, you will receive an automated message confirmation. However, if you feel the need to double check, feel free to contact us and we are happy to ensure your order was received.
Of course, we serve customers from all over the globe. When you set up your online account, leave the “state” blank and ship your samples to 5350 Partners Court, Suite C, Frederick, MD 21703.
Our lab in Frederick is open 24 hours, Monday through Saturday. We are closed for New Year’s Day, President’s Day, Memorial Day, Independence Day, Labor Day, Thanksgiving, and Christmas.
We offer free sample pickups in the Washington DC metropolitan area. Please contact us to find out the schedule by territory.
To properly prepare your samples for sequencing, please follow our guideline here.
To improve the sequencing results of your samples, ensure that all information on the sample sheet is accurate, and include any additional important information (e.g., hairpins or high Tm primers). In the event some reactions fail, providing excess sample can improve the turnaround time for repeat reactions.
We accept plasmids, purified and unpurified PCR, cosmid, and BAC samples. We also accept agar plates with bacterial colonies and bacterial cultures to purify the samples at our lab for an extra cost.
Of course, we encourage the use of strip tubes over individual tubes regardless of the number of samples.
Yes, however we recommend sequencing PCR products that are at least 200 bp.
Our experts will work closely with you to optimize complex GC-rich samples and troubleshoot using proprietary protocols specifically designed for such cases.
We store all samples for one month after sequencing. We also store customer primers free of charge until completely finished.
Please label each strip with your initials, and sample number on the side of each tube. We also recommend labeling the top of the tubes.
Just submit your unpurified PCR products with primers in separate labeled tubes. We will perform the clean-up, run each sample on gel, then optimize and sequence each reaction.
Please look at your vector map or the manufacturer’s procedure to see which primer site is present or which primer is recommended for sequencing. We offer free primers, check here for a list of our primers.
We recommend using gel electrophoresis or a spectrophotometer. If using a spectrophotometer like a NanoDrop, check the A260/A280 to ensure your sample is not contaminated.
We will store all primers you send us free of charge upon request to use for future orders.
It is ideal to design primers that have a GC content of 40-60% and Tm of 50-60oC.
We are happy to work with you to design primers to optimize your sequencing. We will also order your primers for you free of charge from XXXXX; just contact us at [email protected] for more info.
We are working towards synthesizing own custom oligomers. However XXXXXX is a great source for ordering the primers you need.
We offer free primers. Click here for a list of our universal primers.
It depends on the number of reactions the primer is being used for, but we will need at least 1 µL with 30ng/µL or higher concentration per reaction.
Once we receive the samples in our lab, the average turnaround is 8 hours to complete the sequencing. It will depend on your location for the exact data delivery time, but in most cases it will be next day delivery. We also offer an express service if you are in a rush, contact us for more info.
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 attachment, and you will receive a custom sequence summary from a member of our QC team.
The average read length for the sequencing instrument is 800-1000bp. For longer reads ask us about our primer walking service
There are many reasons a reaction could fail. Our QC team will closely analyze your data and suggest the most probable reason for the reactions that failed along with the best course of action such as rerunning the sample or repeating the entire reaction.
We will optimize samples from orders that did not sequence correctly and rerun them later that day. If a reaction needs to be repeated from scratch due to an error on our end, we will do so free of charge. We will ask if you want to rerun any other incomplete reactions.
There are many possibilities for why a reaction fails. Our dedicated QC team will examine each failed reaction to determine the most likely cause and relay the findings in your personalized order summary.
If a DNA sample contains a homo-polymeric region such as poly A, 5 or more in concession, the enzyme can slip from the strand and result in a bad read. Should this be the case, we will recommend sequencing the DNA in the opposite direction to improve results.
It will largely depend on the reason for the poor results. We will recommend the best options for optimization based on our expertise and work closely with you to sequence the most complex reactions.
Please email us your specific order numbers and we will quickly respond to let you know if we still have the sample. We typically store all samples for one month.
Please contact us by phone (1-301-761-4835) or email ([email protected]) if you have any questions about your invoice.
We accept payment by credit card or Purchase Order (PO). Include the PO# for your group or institution in the account info.
We prefer our customers to place their orders online. However, if you need to submit orders manually, please contact your regional account manager (INFO) to arrange a pick up.
If you submit your samples prior to the pick-up deadline, they will be picked up. If you submit your sample after the deadline, it may not have been picked up so please contact our lab and we will gladly check for you if we have received your samples.
If you missed the local drop-off time, we may still be able to pick it up for you. Call or email the local manager (INFO), and we will try our best to arrange a pickup.