SAMPLE SUBMISSION GUIDELINES
APM reserves the right to refuse any sample that does not conform to our conditions. Poorly prepared samples can cause equipment problems and can result in significant costs of both time and money. Pre-submission Consultation
Many researchers who wish to do proteomics projects are unsure of how to proceed and how to best get mass spec. ready samples to the core facility. If you are in this category contact Jack Moore (jmoore@ualberta.ca, 780-492-2522) and he will assess your needs and will either answer your questions or set up a meeting with one of our faculty members to discuss your needs and guide your work. Submitting Samples
If it is the first time that you are using the facility, and you are ready to submit, contact Jack and he will arrange a time for you to meet with him in APM. He will walk you through the process and you can then submit any future samples without his help. If you do drop off samples without a meeting email Jack and give him a quick overview of what you have done and what you are looking for. Shipping Samples
- Ship samples frozen on dry ice.
- email Jack with the tracking number so that he can check up on any delays
- Insert a copy of our sample submission form or email the form to Jack
- ship overnight, FedEx is preferred, to the following address:
University of Alberta - Central Receiving
116 St and 85 Ave
Edmonton, Alberta, Canada
T6G 2R3
Attn: Jack Moore, Biochemistry, 4096 Katz
SAMPLE PREP GUIDELINES
Avoiding Contamination
The presence of some contaminants can be very troublesome and can result in poor, or even no, data. Keratin (dust, skin, hair, etc.), PEG, detergents, high salt concentrations and many polymers are the most common problems that we see. Some tips can be found here.
In-gel Sample Prep
- Supply as much protein as possible. Our instruments are very sensitive but supplying us with as much protein as you can increases the chances of our collecting ideal data.
- Aim for getting as much protein per gel volume as possible. If you have a non-complex sample (less than a thousand expected proteins, IPs, etc.) only run your gel for a cm or so.
- 1mm gel thickness is preferable, the thinner the gel the higher the likelihood of losses during staining and destaining.
- We recommend using Coomassie blue for staining. Remember that the only reason you stain proteomics samples is to visualize them so only stain long enough to accomplish this. Keep an eye on it and when you can see the bands stop the staining.
- Destain thoroughly using standard conditions until there is a clear background with clearly visualized bands.
- If excising your own bands, we will do it if you like, cut as close to the stained area as possible. Exclude any unstained gel.
- If running several lanes, pool all bands of the same protein(s) into a single tube for submission.
- Wash the gel slices twice with 50% acetonitrile in water, discard and add a small amount of water to keep it moist. You do not need to submerge the gel.
- Store frozen at -20 until you submit
MALDI-TOF Sample Prep
- Only use volatile buffers/solvents such as water, methanol, acetonitrile, etc.. Avoid non-volatile salts (NaCl, CaCl2, KH2PO4, etc.), detergents, urea, guanidine, glycerol, DMSO, DMF. If you can’t get away from these remove them after, ziptips and reversed phase LC will often help.
- Sample amounts needed for MALDI are related to the molecular weight of the analyte. We require more concentrated samples for larger molecular weights. Ideal samples are 5uL of a 15 picomoles/uL for smaller MWs (1000 or less), 50 picomoles/uL for 20,000 and 100 picomoles/uL for 75,000. Remember it’s always best to supply as much as you can afford, we have a much better chance of delivering good data with more concentrated samples.
APM reserves the right to refuse any sample that does not conform to our conditions. Poorly prepared samples can cause equipment problems and can result in significant costs of both time and money. Pre-submission Consultation
Many researchers who wish to do proteomics projects are unsure of how to proceed and how to best get mass spec. ready samples to the core facility. If you are in this category contact Jack Moore (jmoore@ualberta.ca, 780-492-2522) and he will assess your needs and will either answer your questions or set up a meeting with one of our faculty members to discuss your needs and guide your work. Submitting Samples
If it is the first time that you are using the facility, and you are ready to submit, contact Jack and he will arrange a time for you to meet with him in APM. He will walk you through the process and you can then submit any future samples without his help. If you do drop off samples without a meeting email Jack and give him a quick overview of what you have done and what you are looking for. Shipping Samples
- Ship samples frozen on dry ice.
- email Jack with the tracking number so that he can check up on any delays
- Insert a copy of our sample submission form or email the form to Jack
- ship overnight, FedEx is preferred, to the following address:
University of Alberta - Central Receiving
116 St and 85 Ave
Edmonton, Alberta, Canada
T6G 2R3
Attn: Jack Moore, Biochemistry, 4096 Katz
SAMPLE PREP GUIDELINES
Avoiding Contamination
The presence of some contaminants can be very troublesome and can result in poor, or even no, data. Keratin (dust, skin, hair, etc.), PEG, detergents, high salt concentrations and many polymers are the most common problems that we see. Some tips can be found here.
In-gel Sample Prep
- Supply as much protein as possible. Our instruments are very sensitive but supplying us with as much protein as you can increases the chances of our collecting ideal data.
- Aim for getting as much protein per gel volume as possible. If you have a non-complex sample (less than a thousand expected proteins, IPs, etc.) only run your gel for a cm or so.
- 1mm gel thickness is preferable, the thinner the gel the higher the likelihood of losses during staining and destaining.
- We recommend using Coomassie blue for staining. Remember that the only reason you stain proteomics samples is to visualize them so only stain long enough to accomplish this. Keep an eye on it and when you can see the bands stop the staining.
- Destain thoroughly using standard conditions until there is a clear background with clearly visualized bands.
- If excising your own bands, we will do it if you like, cut as close to the stained area as possible. Exclude any unstained gel.
- If running several lanes, pool all bands of the same protein(s) into a single tube for submission.
- Wash the gel slices twice with 50% acetonitrile in water, discard and add a small amount of water to keep it moist. You do not need to submerge the gel.
- Store frozen at -20 until you submit
MALDI-TOF Sample Prep
- Only use volatile buffers/solvents such as water, methanol, acetonitrile, etc.. Avoid non-volatile salts (NaCl, CaCl2, KH2PO4, etc.), detergents, urea, guanidine, glycerol, DMSO, DMF. If you can’t get away from these remove them after, ziptips and reversed phase LC will often help.
- Sample amounts needed for MALDI are related to the molecular weight of the analyte. We require more concentrated samples for larger molecular weights. Ideal samples are 5uL of a 15 picomoles/uL for smaller MWs (1000 or less), 50 picomoles/uL for 20,000 and 100 picomoles/uL for 75,000. Remember it’s always best to supply as much as you can afford, we have a much better chance of delivering good data with more concentrated samples.
