Finalize Protein Extraction Protocol

[sr320]

@Ellior2 is getting a markdown version of the protein extraction protocol up at https://github.com/sr320/LabDocs/tree/master/protocols

Once a draft is up lets make sure it is clear to all and we have necessary reagents.

[sr320]

Re incubator - yes there is a big one in the corner lab, maybe a smaller one in main lab. what size tubes?

[kubu4]

@Ellior2 - Actually, we received the trypsin. My fault for not getting it entered on the Purchasing Log. Whoops! Sorry! :disappointed: However, I think I showed you where we stored it - in that drawer in the -20C.

37C incubator is in the lab with the qPCR machine. There are two. Not sure if either of them is currently turned on. I believe one of them has markings indicating the correct dial position to achieve 37C (the older model). The gray one has a dial with numbers and those numbers can be used relatively accurately to achieve the desired temps. Regardless, you'll need to get them turned on and check their temp a couple of times over an hour or two to make sure the temp is stable, prior to actually putting samples in them.

[Ellior2]

@kubu4 Oh I thought you were showing me where you put the Lys-C...but it looks like that is in the freezer. And the Lys-C is only 2ug? Hmmm....

Were we supposed to order 125-05061 ?

[Ellior2]

Nm... I found Emma's remarks on how to make Lys-C aliquots

[kubu4]

I don't know what was supposed to be ordered. I ordered the catalog number @emmats listed in issue #340 .

[sr320]

This should list what was ordered https://docs.google.com/spreadsheets/d/1DHXiiEzWhh0XHnEMvKz4TX10e69ErOWHv5Jo-ouk6x8 On Wed, Nov 23, 2016 at 8:10 PM Rhonda Elliott notifications@github.com wrote:

@kubu4 https://github.com/kubu4 Oh I thought you were showing me where you put the Lys-C...but it looks like that is in the freezer. And the Lys-C is only 2ug? Hmmm....

Were we supposed to order 125-05061 http://www.wako-chem.co.jp/english/labchem/product/life/Lys-C/pdf/print.pdf ?

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[kubu4]

@Ellior2, we did get the trypsin, though. It's in the same freezer. Possibly even in the same location as the Lys-c.

[Ellior2]

@sr320 Tubes will be 1ml snaptop centrifuge tubes

@emmats when I prepare the Lys-C, do the leftover 20ul aliquots I make belong in the -20C, -80C or neither? What is HPLC grade H20? Is that even more special than nanopure?

Alright I'm done for the night. No more questions lol! Happy Thanksgiving ya'll!

[emmats]

For 5 of my samples, I have less than 1ug/1ul of protein, which means that I will have less than 100ug of protein for steps 1-8. For the remaining samples in which I had more than 1ug/ul I am diluting so I have exactly 100ug of protein in the 100ul required starting volume. So at Step 9, can I continue when some of my samples have less than 100ug? Do I just adjust the amount of LysC and trypsin?

Why do you have so little sample? You had plenty of tissue, right? If you have more tissue, then you need to go back to your original samples and get 100 ug, I think that is the best choice. If you don't have time for that, then start the digestions with the same amount of protein per sample (so go with the lowest amount). You will have to adjust your enzyme amounts.

Is the process for adding Lys-C the same as the Trypsin process that you described in more detail since they are both (1:30 enzyme:protein).

I'm not sure what you mean. You add Lys-C as described in the protocol and incubate for 1 hour.

From your notes: Prepare the number of trypsin bottles needed (you will want 5 µg of trypsin for each sample, or 1 µg trypsin: 20 µg protein). Add 20 µl water or trypsin buffer to each bottle of trypsin and vortex lightly. Aliquot 5 µl of trypsin to each sample for 100 µg of sample (1:30 enzyme:protein).

But is it a 1:30 ratio or a 1:20?

Oops - I always do the latter (1:30). I think both will get you good digestion. There is a paper on trypsin amounts and incubate time that I can dig up for you if you're interested in knowing more.

For the trypsin, I calculated how much I will need based off of the amount of protein in my samples and it looks like I will need 105.4ug so we are 5.4ug short. Can we borrow some from your lab and order more? If I am correct in assuming you add Lys-C in the same concentrations as you would the trypsin, then I would also need 105.4ug, again possibly 5.4ug short. We also haven't received our trypsin and don't know if we will by Monday. It's the last thing we need.

Sounds like you are ok for trypsin, although @kubu4 overbought for us once so you can always borrow some if you are short. If you do not have Lys-C I can bring some aliquots Monday.

[Ellior2]

@emmats So I definitely have enough protein in the total sample I have sonicated- the problem is that it is less concentrated than 100ug/100ul. So in order to have 100ug of protein for the digestion, I would have to start out with 120-130ul instead of 100ul. The lowest concentration I have is 75.5ug/100ul which means I would have to bring all the samples down to 75.5ug starting protein amount.

I will go ahead and do the 1:30 ratio for the trypsin if that is what we did for the 2015 samples. If we do a 1:30 ratio we will only need 2.5ug trypsin for 75.5ug protein/sample for a total of 55ug for all 22 samples.

From your lab notebook "Made aliquots of LysC (from Wako) following Villen lab protocol (see attached).  Added 450 µl HPLC grade H2O to 2 AU vial.  Made 20 µl (40 µg) aliquots at concentration of 2 µg/µl.  Will add 0.5 µl to each 100 µg sample."

Looks like you are adding 1ug of LysC for every 100ug of sample, hence a 1:100 ratio. In the protocol however it says 1:30...?

I may need assistance getting the incubator fired up in the morning.

[emmats]

You should evaporate the samples a bit and then bring the volume back up to 100 µl. Then you will have 100 µg in 100 µl. I'm not sure why your samples have relatively little protein in them. It sounds like you lost a lot and/or did not lyse cells well during the homogenization/sonication. When we did this before we had closer to 2 ug/ul.

Remember, all the details for what we did previously are here: https://www.evernote.com/l/APLBpNYN-VlFvbPyRfi7DbOQX3q2u6s2mbQ Just follow that for enzyme amounts.

I think I adjusted the Lys C protocol from what the Villen lab does. I now add 3 ug per sample.

I have a 10 am meeting on Monday, but should be able to get to the Roberts lab before 9 to help you get set up. Is that ok?

[Ellior2]

Ahhh! I forgot to account for the 22ul of bicarb I added. That should make it better. Let me rework it!

[Ellior2]

This is more like it see table

Ok so 3ug of Lys-C per sample and 3ug of Trypsin per sample.

[Ellior2]

@kubu4

Trypsin is not with the Lys-C and no where obvious (to me) in the freezer.

[kubu4]

I'll come by in a few minutes to help.

On Mon, Nov 28, 2016, 08:03 Rhonda Elliott notifications@github.com wrote:

@kubu4 https://github.com/kubu4

Trypsin is not with the Lys-C and no where obvious (to me) in the freezer.

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[Ellior2]

@kubu4 Did the methanol make it onto the Mychem sheet?

[kubu4]

Doesn't look like it. Feel free to add it, if you're so inclined.

[Ellior2]

@emmats I forgot to order autosampler vials for the MS. Can I "borrow" 22 for tomorrow?

[Ellior2]

@emmats

Here are the desalting steps that I wrote up. Would you mind looking through it and making sure that I have it right- paying special attention to what I am discarding and where my peptides are during each step?

[emmats]

Just bring your samples in tubes down to UWPR on Monday and we can use their autosampler vials. They are included in the MS pricing.

Desalting Step 2 - You can discard the liquid at the bottom of the columns every other addition of solvent A (it easily fits 400 ul at the bottom)

Step 5 - I always keep the salt fraction in case something goes wrong. I did have to go back to my salt fraction once when I used the wrong desalting columns.

Step 8 - this should be 0.1% formic acid! Thanks for finding that typo in my protocol.

[kubu4]

A couple of things to add to this:

• The spin steps in @Ellior2 desalting protocol are listed in RPMs instead of RCF. RPMs result in different RCF depending on which centrifuge is used. For example, we have three different centrifuges in the lab. If you spun the same sample at 2000RPM in each of those centrifuges, each centrifuge would apply a different amount of force (RCF) to the sample.

As such, if utilizing RPMs, the centrifuge model should be listed. Otherwise, use RCF instead, which can be easily set, no matter which centrifuge you're using (the centrifuge will spin at the correct RPM to achieve the desired force).

• The columns are on the shelf with all the kits in FTR213. They're in a big plastic bag.

[emmats]

@kubu4 the exact speed/force doesn't matter that much, as long as the liquid is pushed through.

[kubu4]

@emmats In this particular instance, sure. However, for other applications requiring a centrifuge, it's important information. So, it's only proper to be consistent; for reproducibility sake.

[Ellior2]

@emmats I made the solvents for desalting taking extra care to minimize plastic contamination and stored them in glass containers. Now that I have them made, can I pipette directly out of my working bottle since it is more dilute or should I keep pouring off little amounts into beakers to pipette from? This would end up adding quite a lot of time to the procedure and produce a lot more waste, but I'll do it if it's a concern.

[emmats]

That is fine. Although I usually make smaller bottles (100-200 mL) to pipette from, which is generally a good practice. You don't want to pipette directly from large bottles of reagents because you could easily contaminate the whole jar.

[sr320]

@Ellior2 is it safe to say that the knowledge from this 75+ commented issue has been incorporated into?

https://github.com/sr320/LabDocs/blob/master/protocols/ProteinprepforMSMS.md

[Ellior2]

@emmats Thanks, I had only made 150 ml of each reagent.

@sr320 It is documented pretty well in my online lab notebook, but I will work on incorporating it all into the master protocol. It will probably end up becoming a 10 page document if that is what you want.

[Ellior2]

Whew! I finished! This thread is almost over I promise...

One more question for @emmats : During some steps you explicitly say to vortex and some you do not. For example, it is stated in the digestion protocol to vortex after the DTT addition but not after the IAA addition. Is there reason for this? Could vortexing when it's not needed potentially ruin your sample?

[emmats]

Assume vortex when it seems logical. It will not ruin your sample.

[Ellior2]

For @yaaminiv and @laurahspencer coming up on your extractions- I believe all you need to order is more trypsin.

[kubu4]

Bringing this back from the dead. 💀

I think the protocol should list vendor and catalog numbers for stock reagents.

I also think it would be helpful to list needed equipment at the beginning of the protocol. Possibly indicate if equipment is available in Roberts Lab or if it's externally accessible (and where/who's lab).

[Ellior2]

Alright, I think I've added most of the necessary equipment. Feel free to add and update @laurahspencer @yaaminiv

[Ellior2]

I added the things we recently bought and listed the vendors and catalog numbers. I searched for some of the ones we already had in the purchasing log- couldn't find them. I looked them up on Mychem and added the quantity we bought and from what vendor. I could not find the cat. # on those however.