Orbi is running Valerie’s samples today, after which Orbi will be cleaned up and ready for the next week. The facility will be closed for Thanksgiving during November 28 and 29, so there will be no access to the open access instrument and plate reader.
I would like to share with you a resource for custom peptides and other life sciences research products that might save you a few dollars. As always, a disclaimer: I don’t promote any products or services on this blog and I don’t accept any form of compensation for sharing this information.
The following is contributed by Dr. Ann McPherson, Territory Sales Associate Biotech/Pharmaceutical Accounts Specialist for Bio Basic
“Bio Basic is a one-stop-shop for customers doing research in the life sciences field. We distinguish ourselves from other one-stop-shops in three main ways:
Manufacturing. We have ten product lines, eight of which are manufactured within our state-of-the-art facility.
Lowest price for all product lines. By directly manufacturing most of our products, we’re able to offer the lowest prices to our customers.
Strict quality control. All of our products are Quality Control tested before shipping. Bio Basic is also ISO9001 certified, which means that we’re regularly audited for third party assessment purposes that certify that we adhere to the method and model of quality management published by the International Organization for Standardization.
Information on our products and services can be found on our website. If you e-mail me (email@example.com) with the specific products and services that you’re interested in, I can provide more information and may be able to offer an additional discount. For now, I would like to make users of Penn State’s Proteomics and Mass Spectrometry Core Facility aware of two promotions that we have until Dec. 31st, 2013:
“Buy 2 get 1 FREE!” on all of our products (excludes our services: Gene Synthesis, Oligo Synthesis, DNA Sequencing, Peptide Synthesis, Protein Purification, Antibody Services). Use the quote number FBG2013ANN when ordering.
30% off of all recombinant proteins! Use the quote number PRTN30ANN when ordering (minimum $45 order).
You can find out about our range of recombinant proteins here.
For various cytokines, look under the chemokine, interleukin, interferon, tumor necrosis factor, colony stimulatory factor (CSF), etc. categories.”
If you have never done in-gel protein digestion, this tutorial is for you! The protocol is really simple and does not require any specialized equipment. Even if your gel has been sitting in the fridge for a month or two, it should still work, but no mold, please! First order of business is to choose a band and cut it out. You will need a clean blade, a clean surface (a clean glass plate or a clean transparency sheet), a clean microcentrifuge tube, and a pair of clean gloves. Everything should be clean: the goal is to minimize contamination of interesting proteins by uninteresting keratins. 1. Place the gel on a glass plate, blot excess water with a clean paper tissue. Select a band and cut out only the stained portion of the gel. Avoid unstained area: it will sponge up a protease solution, giving nothing interesting in return. 2. Dice the band into 1-mm (1/16”) sections; this will help the protease reach more protein inside the gel. 3. Place the gel cubes in a clean microcentrifuge tube, cover with water to prevent them from drying out, and label the tube with no more than 5 characters. For example, use your initials and numbers. 4. Include a positive control! There is no charge for analyzing a control! Cut out a known protein band following steps 1, 2, and 3.
The in-gel digestion protocol can be found on Thermo website along with the product numbers for all necessary reagents.
Washing the gel
You will need 50% acetonitrile (ACN) solution containing 8 mg/mL ammonium bicarbonate (wash solution). Add 0.05 – 0.1 mL of this solution per gel band, enough to completely cover the gel. Incubate at 37 C for 15-20 min, discard the solution. Repeat two more times. At this point, all or most of the blue color should be gone. If you are destaining SYPRO, there is no easy way to tell whether it is all gone, of course.
Reducing disulfides and alkylating Cys residues
You will need
- 5 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) solution in the 8 mg/mL ammonium bicarbonate. I make 100 mM (29 mg/mL) stock solution in water and store it in the fridge up to a month. Dilute this stock 20x (e.g. 50 uL 100 mM TCEP + 950 uL ammonium bicarbonate solution).
- Fresh 100 mM IAA (iodoacetamide) solution in the 8 mg/mL ammonium bicarbonate. This is approximately 18 mg/mL IAA solution.
Cover the gel slices with 5 mM TCEP and incubate 10 min at 60 C. Remove the TCEP solution and cover the gel with 100 mM IAA solution. Incubate at 37 C for 15 min with occasional shaking, preferably protected from light. Room temperature will also work, but give it extra 10-15 min. Discard the IAA solution and wash the gel with the wash solution three times to remove IAA and TCEP. (In the Pierce protocol, you might notice that 100 mM IAA requires 9.3 mg in 1 mL, while MW of IAA is 184.9. It is a typo. Either way: 50 mM, 9.3 mg/mL or 100 mM, 18 mg/mL, will work as there will be a large molar excess of IAA compared to Cys. Shrink the gel by covering it with ACN and incubating at r.t. for 15 min or until the gel turns white and brittle. Remove ACN and allow the gel to air dry for 15 min at 37 C (or longer at room temperature).
Make 1 mg/mL trypsin stock solution in 50 mM acetic acid or 1 mM hydrochloric acid. This solution can be aliquoted and stored in a freezer for months. Instructions for other MS-grade proteases can be found here. Dilute trypsin to 0.01 mg/ml (1:100 dilution) with 8 mg/mL ammonium bicarbonate. Add 50 uL of this solution per gel band and incubate at 37 C 8-24 hours. If you are working with a large piece of gel or several bands combined in one tube, make sure that after the gel re-hydrates in the enzyme solution, it is completely covered. Add more enzyme solution if necessary.
Extracting the peptides
You will need 50% ACN solution containing 0.1% formic acid (FA). If you don’t have formic acid in your lab, let me know – we will share ours with you. Transfer the digest solution to a new tube. Extract each gel band with 50 uL of 50% ACN/0.1% FA (more, if working with a large volume of gel) by incubating at 37 C for 15 min. Transfer this solution to the new tube with the digest solution. Repeat 2 more times. Evaporate the combined extracts in a vacuum concentrator (e.g. Speedvac). Please note, if you are extracting large peptides (>5000 Da), using a sonic bath may be a better option than incubation. Also, in addition to the three 50% ACN extractions, you can use 100% acetonitrile for the final extraction. Submit your dried samples along with a picture of the gel so that I can estimate how much of each sample to use for analysis, and don’t forget to fill out the Protein ID Request form
As always, let me know if you have a question or three!
Jingjie’s samples are running, next is Mustafa’s in-gel.
Today is one of those days when nothing goes as planned. Oh well. Different plan: washing out chymotrypsin overnight. Everything is delayed one day…
Doug’s new samples are running, next are Jingjie’s samples and then Mustafa’s. We like it when it’s busy!
After my standard, Orbi will be running 3 samples for Doug W. overnight. Eric’s samples are next in the queue.
If someone told me “I honestly believe, the instrument is calibrated, it seemed okay last year when I checked it last time. It looks like the mobile phase is a bit old, I am pretty sure I replaced it 3 months ago when a spider fell into the bottle, so it should work. It is recommended to clean the ion source daily, but who has time, let’s hope it is not too dirty. I’m just going to run your samples and see what happens. Fingers crossed!” chances are, I would take my samples to be analyzed elsewhere.
So when I hear “We used old reagents, but they should still work, right? I found this old trypsin in our lab, I hope it is still okay. I am pretty sure the protein is still there, but it might have degraded. We bought new co-IP beads and are unsure how much protein we have loaded, but fingers crossed – everything worked!” I lose my mojo. If crossing fingers is a part of the experimental design, how are we going to troubleshoot when something goes wrong? Was it old trypsin or was the hypothesis no good?
Good news! There is a way to check if the old reagents still work and if the beads have the expected capacity – run a pilot experiment using something that always works; run a control experiment (or two) in parallel with the real experiment. Carry your control through the same procedure as your unknown. Check every step in the protocol: did it work? Our facility loves controls so much that there is no additional charge for analyzing them. So go ahead, bring as many controls as you think are necessary and you won’t have to cross your fingers!
Orbi is running a couple of standards to make sure the sensitivity is up to specs. After that – Doug’s Cys-modified set of samples.