Biomolecule Analysis Core Facility
The Biomolecule Analysis Core Facility (BACF) contributes to the BBRC by specializing in the separation and characterization of biomolecules. The BACF is available to all BBRC investigators, other UTEP users and external collaborators.
Most instruments are accessible as shared-resources at no charge for the users. Service is also provided for sequencing and identification of proteins and peptides using LC/MS-MS on a recharge basis.
MS sample submission: Login to MS LIMS
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Acknowledgement of the RCMI grant in publications and presentations
Users of the BACF are requested to include the following statement in all manuscripts submitted for publication or meeting abstracts:
We would like to thank the staff of the Biomolecule Analysis Core Facility (BACF) for services and facilities provided. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIMHD or NIH.
NATIONAL INSTITUTE ON MINORITY HEALTH AND HEALTH DISPARITIES (NIMHD) Grant no. 5G12MD007592
New users must undergo a workshop/training session given by the coordinator before using any shared (open access) equipment. Training session(s) should be scheduled with the coordinator (Yanira Cordero, BS) by telephone (915-747-6875 ) or e-mail ( firstname.lastname@example.org ).
Services and Fees
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How to submit MS Samples
How to Submit Samples for MS analysis: Frequently Asked Questions
(NOTE: Items A-E below were reproduced, modified or adapted from Tufts University HPLC/MS Core Facility; http://www.tucf.org/hplc-ms-f.html )
A. How much sample do I need for molecular mass determination or peptide sequencing?
For molecular mass determination, one needs approx. 1-10 picomoles of a purified peptide or protein. If the sample contains non-volatile salt, Sep-Pak C18 purification needs to be performed prior to MS analysis.
For peptide and protein sequencing and identification from SDS-PAGE gel bands, any band visible by Coomassie staining is acceptable (roughly corresponding to a minimum peptide or protein concentration in the range of 1-0.1 pmol/µl). You may also combine up to 3 duplicate gel bands in one single microtube to send us for analysis. This way you will increase your chances to get one or more peptide sequences and you're your protein identified.
B. Sample Preparation:
Human keratin is a very common contaminant!!! Therefore, the following extra measures must be taken to minimize the contamination:
Wear gloves at all times during sample preparation. Wash outside of gloves with water before using them to handle your samples and vials. Even powder-free gloves still contain powder, which eventually contaminates samples.
Thoroughly wash anything that will come into contact with your sample (i.e. gel apparatus, staining trays, gel excising implements, gel storage equipment, and Eppendorf vials) to remove human keratins and other contaminants. Microtubes should be washed with HPLC-grade acetonitrile and then HPLC-grade water. Sample microtubes should be free of plasticizers and other common contaminants (e.g., detergents, synthetic polymers) that can interfere in highly sensitive mass spectrometric analysis. Anything that touches the gel or sample is a possible source of contamination. Avoid storing gel in saran wrap or similar material and instead use new, cleaned plastic or glass gel trays.
Avoid using molecular weight cutoff filters (e.g., Centricon filters) and if they must be used, thoroughly wash them. We find these are often a source of synthetic polymer contamination in samples.
If you have a complex sample (purified/isolated from blood or plasma, or whole tissues or organs; or from cells maintained in fetal bovine serum, albumin, or any other non-defined medium), please call or e-mail the core director (Dr Igor Almeida, 915-747-6086; email@example.com ) to discuss your sample, before shipping it to us.
Avoid using surfactants and detergents (i.e., CHAPS, DMSO, Triton-X, NP-40, etc.). If these must be used please contact the core director to discuss suitable concentrations. Also, try to minimize salt and buffer concentration in the sample, particularly non-volatile salts.
Vials: For liquid samples use the smallest vial necessary, (as low as to the 0.5-0.6ml vials). For gel samples, use a washed 1.5 ml Eppendorf tube (the vial you send us will be used for the in-gel digestion procedure so please make sure it is clean). Don't use vials with rubber o-rings or gasket. Avoid colored vials and avoid using paraflim to seal vials.
Vial label: each of the vials that contain your samples must be labeled with the your name, sample name (choose something unique but short and different than sample name of previously submitted samples), and date. Please label both top and side of vial.
After the gel has been run, non-specific dust contamination can still be introduced so keep samples covered or protected as much as possible.
All samples must be shipped with completed sample submission forms.
C. Gel and staining suggestions
Use standard SDS-PAGE or 2D gels. Maximize the amount of protein on as minimal amount of gel as possible. A concentrated gel band works a lot better than a large diffuse gel band.
Gel thickness of 1mm is preferred. Gradient, denaturing and native gels are acceptable.
Stain with Coomassie Blue using standard conditions. Stain only until your bands are visible. If the amount of your protein is too low for coomassie stains, use silver or Sypro stains. Generally we recommend Sypro stains (see: http://www.lifetechnologies.com/us/en/home/brands/molecular-probes.html or http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Proteomics_and_Protein_Expr/Protein_Analysis/Protein_Electrophoresis/SYPRO_Protein_Detection_Reagents.html ) over silver stains due to higher compatibility with mass spectrometry Sypro stains require a UV source to visualize gel spots or bands. If silver stain must be used, we require you use the Invitrogen Silverquest stain kit ( http://www.invitrogen.com/content/sfs/brochures/713_021258_SlvrQst_bro.pdf ) as we find other silver stains incompatible. Silver stained gel spots or bands have to be totally destained as the silver has been reported to inhibit trypsin digestion of proteins.
D. How to cut gel bands and prepare samples to be sent to us
Destain gels thoroughly. A clear background is preferred. All excess stain must be removed otherwise residual stain artifacts may interfere with our data acquisition.
Excise band as precise as possible. If there is any diffuse stained edges to the band, omit it and excise only the clearly stained band.
Excise a band from a blank area of gel, about the same size as your sample gel bands (cut in the step above), and send it to us to run as a gel blank. You will not be charged for analyzing this gel blank. Add this as a sample on the sample submission form and label it 'gel blank.'
Place each gel slice into a washed, plain 1.5 ml Eppendorf tube. Do not use any tubes with O-rings or gaskets.
Wash the gel slice with 50% HPLC-Grade acetonitrile/Water at least 2x for 10 min with occasional vortex mixing. Discard the wash solution. Use plastic pipette tips to remove the solution as the gel slice tends to stick to glass Pasteur pipets. This step helps wash out any residual detergent or salt.
Rinse the Eppendorf cap off with the 50% HPLC Grade Acetonitrile/Water solution and close the cap tightly. No additional solution is needed to cover the gel band. Do not parafilm the tube. The gel band will remain moist and can be stored in a -20 degree freezer or shipped to us. Ship the gel band to us on dry ice, if this is not possible please contact us to discuss. Put your samples in a bag instead of loose in the dry ice as vial tops can come loose. For local users, please try to drop off your samples in the early morning as this is when the in-gel digestion procedure for the day begins.
E. What address do I send my samples to?
Biomolecule Analysis Core Facility
Attn: Dr Igor C Almeida
Department of Biological Sciences
University of Texas at El Paso (UTEP)
500 W University Ave., Bioscience Bldg., room 5.208
El Paso, TX, 79968
Phone: (915) 747-6875; Fax: (915)-747-5808;
F. Where can I find the sample submission form?
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