Posts in ABRF Discussion Forum

Adding to the list of why Covid-19 is more serious than the flu.
Speaking from personal experience after many flu infections, my life best I can
tell went on as normal after recovery.  We are only now learning that is not
necessarily the case with Covid-19 survivors.  Impaired liver and kidney
function as well as scarring of heart tissue are the more serious of the post
infection damage a recovered patient will be living with.

See https://coronavirus.nautil.us/what-are-the-long-term-effects-of-covid-19/
with sources listed in the article.

Best regards,
Brian

Rest of post

________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of chitty
<email obscured>>
Sent: Tuesday, March 31, 2020 5:00 PM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] Requesting help with COVID-19 information
response on social media

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Yes, that was a good video.

I want to mention that I got some good advice from Isabelle Girard, which I
want to share. It was very helpful to me in recent conversations. Here is what
she had to say:

It is tempting, as scientists, to respond to such a post with a detailed
argument about numbers and statistics.  As many have pointed out, I think this
generally leads only to an argument about predictions, which are not facts
(yet) for either side.  So here are my thoughts about a response:

Dear Guy,

You ask a very relevant question.  Is our response appropriate to the threat
that coronavirus poses, given that we respond differently to influenza?  This
is exactly the question that epidemiologists must answer, given that they did
not promote such measures in the U.S. for HIV, Ebola or Zika.

There is a lot of evidence that this coronavirus is much more serious than the
seasonal flu, although the seasonal flu can be very serious.  During severe
outbreaks of flu, we respond aggressively by getting vaccinated, staying home
when we are sick, and occasionally closing targeted locations like schools and
workplaces.

You are right that the fatality rate for the virus is not YET as high as the
seasonal flu, but we recognize that we are only in the early phases of the
epidemic.  Here's why I think we should take the situation very seriously and
why the drastic measures for social isolation are warranted:

a.  it's more contagious than the flu
b.  it has a longer incubation period, allowing for more spread
c.  the mortality rate is much higher
d.  no one has immunity
e.  there are no vaccines or effective treatments
f.   most people who need ventilators do not survive

If you are correct, and this new virus fits the pattern of seasonal influenza,
we should very rapidly see a flattening out of the infection rate in affected
areas in the next week or two.  In such a scenario, I have no doubt that
leadership will allow a return to more normal operations and our economy will
quickly rebound.  If the infection rate keeps rising, however, it indicates
that we are not dealing with a flu-like disease but something much worse.  We
should stay the course and keep ourselves and our neighbors as safe as we can.

This approach that Isabelle suggested helped me keep the focus on the things we
do know, rather than the slippery numbers discussion. Then, for those who want
to join in a real discussion about it, I engage further.

――
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Hello

We frequently analyse 300 genotypes  using HPLC in our plant breeding programs
using Agilent open labs version C. We would like to merge all files from all
300 runs into one file so we can rank, them graph them etc. I see there is
cross summary sequence reports but this doesnt merge those files

Is anyone aware of videos or PDFs how to do this??

Thank you

Rest of post

Peter

Wow.

At that throughput cleaning/disinfecting and maintaining should be considered
as part of package/system. Mentally walking through the steps - including
cleaning waste disp.

Matt

> On Apr 23, 2020, at 12:24 PM, <email obscured> wrote:
>
> Thanks to everyone who replied.
>
> To clarify, I am looking for something to help us dispense liquid into
100,000 15 ml conical tubes / week.  Found a fancy peristaltic “filling” unit
that will take us to the next level … but we will look at other suggestions as
well.
>
>
https://www.watson-marlow.com/us-en/range/flexicon/tabletop-filling/pf7-aseptic-liquid-filler/

Rest of post

>
> Stay well everyone.
>
> Frances
>
> ZRC 1563
> Office     646-888-2354
> Mobile   516-967-0656
>
>
> From: Frances Weis-Garcia <email obscured>>
> Date: Thursday, April 23, 2020 at 1:27 PM
> To: ABRF Discussion Forum <email obscured>>
> Subject: Large Volume Sterile Liquid Handling
>
> Is anyone aware of a piece of equipment that can fit inside a tissue culture
hood and dispense liquid in a sterile manner into 15 ml conical tubes?
>
> Frances
>
>
> Frances Weis-Garcia, Ph.D.
>
> Head
> Bi-Institutional Antibody and Bioresource Core Facility
>
> Associate Laboratory Member
> Immunology Program
>
> Memorial Sloan Kettering Cancer Center
> 1275 York Avenue, New York, NY 10065
> T 646.888.2354   C 516.967.0656
>
> ABCF-MSKCC
website<https://www.mskcc.org/research-advantage/core-facilities/monoclonal-antibody-core-facility>
>
> Adjunct Faculty
> Center for Clinical and Translational Science
>
> The Rockefeller University
> 1230 York Avenue, New York, NY 10065
> T 212.327.7030
>
> ABCF-RU website<https://www.rockefeller.edu/monoclonal/>
>
>
> Please note that this e-mail and any files transmitted with it may be
privileged, confidential, and protected from disclosure under applicable law.
If the reader of this message is not the intended recipient, or an employee or
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the sender immediately by replying to this message and deleting this message,
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>
>
> =====================================================================
>
>     Please note that this e-mail and any files transmitted from
>     Memorial Sloan Kettering Cancer Center may be privileged, confidential,
>     and protected from disclosure under applicable law. If the reader of
>     this message is not the intended recipient, or an employee or agent
>     responsible for delivering this message to the intended recipient,
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>     copying, or other use of this communication or any of its attachments
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>

Thanks for all the suggestions. Edman sequencing unfortunately is not an
option for this one. And looks like CNBr will be the way to go...

Rest of post

Yan

On Thu, Apr 23, 2020 at 2:26 AM mortz <email obscured>> wrote:

> Hi Yan
> You could actually try N-terminal Edman sequencing :-)
>
> Each sequencing cycle gives the HPLC chromatogram of each cleaved residue,
> and the peak area gives semi-quantitative information.
> If you you cannot separate the 3 forms, and you know the protein sequence
> you can look at the yields of the 2. and 3. residue after M. to get
> relative amounts of each peptide.
> If you can separate the forms, you can also look at M amounts for the 1.
> residue.
>
> This analysis will require sufficient material of each form, ie 30-100pmol.
> Br
> Ejvind
> ――
> View topic http://list.abrf.org/r/topic/19hAp9GquNj5q3evvDD0We
> Leave group <email obscured>?Subject=Unsubscribe
>
>

Thanks to everyone who replied.

To clarify, I am looking for something to help us dispense liquid into 100,000
15 ml conical tubes / week.  Found a fancy peristaltic “filling” unit that will
take us to the next level … but we will look at other suggestions as well.

https://www.watson-marlow.com/us-en/range/flexicon/tabletop-filling/pf7-aseptic-liquid-filler/

Stay well everyone.

Frances

ZRC 1563
Office     646-888-2354
Mobile   516-967-0656


From: Frances Weis-Garcia <email obscured>>
Date: Thursday, April 23, 2020 at 1:27 PM
To: ABRF Discussion Forum <email obscured>>
Subject: Large Volume Sterile Liquid Handling

Is anyone aware of a piece of equipment that can fit inside a tissue culture
hood and dispense liquid in a sterile manner into 15 ml conical tubes?

Frances


Frances Weis-Garcia, Ph.D.

Head
Bi-Institutional Antibody and Bioresource Core Facility

Associate Laboratory Member
Immunology Program

Memorial Sloan Kettering Cancer Center
1275 York Avenue, New York, NY 10065
T 646.888.2354   C 516.967.0656

ABCF-MSKCC
website<https://www.mskcc.org/research-advantage/core-facilities/monoclonal-antibody-core-facility>

Adjunct Faculty
Center for Clinical and Translational Science

The Rockefeller University
1230 York Avenue, New York, NY 10065
T 212.327.7030

ABCF-RU website<https://www.rockefeller.edu/monoclonal/>


Please note that this e-mail and any files transmitted with it may be
privileged, confidential, and protected from disclosure under applicable law.
If the reader of this message is not the intended recipient, or an employee or
agent responsible for delivering this message to the intended recipient, you
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There are bottle top dispensers that can do that. Many different vendors.

See for instance eppendorf's 4966000053.

D. Eric Anderson
Mass Spectrometry Facility, NIDDK
NIH


Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
(301)-496-7546

Rest of post

________________________________________
From: <email obscured> <email obscured>>
Sent: 23 April 2020 13:27
To: <email obscured>
Subject: [ABRF Discussion Forum] Large Volume Sterile Liquid Handling

Is anyone aware of a piece of equipment that can fit inside a tissue culture
hood and dispense liquid in a sterile manner into 15 ml conical tubes?

Frances

Frances Weis-Garcia, Ph.D.

Head
Bi-Institutional Antibody and Bioresource Core Facility

Associate Laboratory Member
Immunology Program

Memorial Sloan Kettering Cancer Center
1275 York Avenue, New York, NY 10065
T 646.888.2354   C 516.967.0656

ABCF-MSKCC
website<https://www.mskcc.org/research-advantage/core-facilities/monoclonal-antibody-core-facility>

Adjunct Faculty
Center for Clinical and Translational Science

The Rockefeller University
1230 York Avenue, New York, NY 10065
T 212.327.7030

ABCF-RU website<https://www.rockefeller.edu/monoclonal/>

Please note that this e-mail and any files transmitted with it may be
privileged, confidential, and protected from disclosure under applicable law.
If the reader of this message is not the intended recipient, or an employee or
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the sender immediately by replying to this message and deleting this message,
any attachments, and all copies and backups from your computer.

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Is anyone aware of a piece of equipment that can fit inside a tissue culture
hood and dispense liquid in a sterile manner into 15 ml conical tubes?

Frances


Frances Weis-Garcia, Ph.D.

Head
Bi-Institutional Antibody and Bioresource Core Facility

Associate Laboratory Member
Immunology Program

Memorial Sloan Kettering Cancer Center
1275 York Avenue, New York, NY 10065
T 646.888.2354   C 516.967.0656

ABCF-MSKCC
website<https://www.mskcc.org/research-advantage/core-facilities/monoclonal-antibody-core-facility>

Adjunct Faculty
Center for Clinical and Translational Science

The Rockefeller University
1230 York Avenue, New York, NY 10065
T 212.327.7030

ABCF-RU website<https://www.rockefeller.edu/monoclonal/>


Please note that this e-mail and any files transmitted with it may be
privileged, confidential, and protected from disclosure under applicable law.
If the reader of this message is not the intended recipient, or an employee or
agent responsible for delivering this message to the intended recipient, you
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Rest of post


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Hi Yan
You could actually try N-terminal Edman sequencing :-)

Each sequencing cycle gives the HPLC chromatogram of each cleaved residue, and
the peak area gives semi-quantitative information.
If you you cannot separate the 3 forms, and you know the protein sequence you
can look at the yields of the 2. and 3. residue after M. to get relative
amounts of each peptide.
If you can separate the forms, you can also look at M amounts for the 1.
residue.

This analysis will require sufficient material of each form, ie 30-100pmol.
Br
Ejvind

Hi Joel,
Since it involves few steps and you do not have to transfer materials, I
would take the peptide solutions and reduce the volume of TFA to a small
droplet and, as you mentioned in your e-mail above, repeat the reduction
and alklation.  Before using the reagents on your precious peptides, I
would test the reduction and alkylation reagents on a standard peptide like
insulin.

If you could check the pH of the peptide solution after adding the R/A
reagents (use a 10uL pipette and pH stips to avoid consuming too much
peptide), and make sure it is no longer acidic, that is a good practice.

After the reduction and alkylation step, you could purify the peptides with
standard Stage-Tip step.
Good luck,
Hediye

Rest of post

On Sun, Apr 19, 2020 at 2:18 AM joel.steele <email obscured>> wrote:

> Hello Everyone,
>
> I have priceless samples that have been processed through an SP3-StageTip
> protocol, I have realised that the TCEP for a reason TBD has failed to
> reduce my samples. I still have the whole peptide sample frozen in 10%TFA
> (loading conditions for Stagetip).
>
> My question is whether I should neutralise the TFA then reduce and
> alkylate in the same vial.
> OR
> Stage tip the sample -> R&A -> stagetip again
> OR
> Abandon sample as the lack of reduction alkylation has possibly led to
> inefficient trypsin digestion
>
> Many thanks
> Joel Steele
>
>
> N.B, On analysis the peptide statistics are higher than that of a normal
> shotgun experiment with reasonable coverage of the proteome.
>
> Side question would this be an intra-protein crosslinking experiment of
> some kind?
>
> ――
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>
>

Hello Yan,

Another option would be chemical cleavage with formic acid which cleaves
predominately at the C-terminal side of Asp, but to some extent also N-terminal
too.  Depending on the distance of the first Asp from the N-terminus of your
protein you could think of this as a "middle down" approach.  This paper
describes some work using formic acid cleavage which will give you a good feel
for what to expect with this type of cleavage.
https://onlinelibrary.wiley.com/doi/full/10.1002/jms.1871  In this paper they
did observe some formylation.

Also, many proteins become acetylated after cleavage of the initiator
methionine. The frequency of this is related to the size of the AA side chain
of the AA following the Met, the smaller ones tend to be acetylated more
frequently than proteins with large AA side chains in the 2- position.

Best regards,
Brian

On 4/22/20, 10:23 AM, "ABRF Discussion Forum on behalf of Yan Wang"
<email obscured> on behalf of <email obscured>> wrote:

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    Thanks Matt and Joel.
    In this case, I'm actually trying to quantify 3 isoforms of the same
    protein starting at different M. So the only thing unique for each would be
    the N-terminal peptide. Two of the 3 would lose the initial M but the 3rd
    one doesn't. Trypsin, ArgC, LysC and alike won't work either as one of them
    have "KKR" at 3-5 position. We are settling with GluC, which only works in
    solution and is not as reliable. With that we can detect 2 out of the 3 in
    cell culture. Now we are thinking if N-terminal peptide enrichment would
    help. I'd like to avoid using CNBr if possible.

    Yan

    On Wed, Apr 22, 2020 at 12:17 AM matt sweeney <email obscured>>
    wrote:

    > One can use intact mass measured compared to predicted if you know
    > sequence.
    >
    > Or one can digest the sample and then obtain the mass of the predicted
    > N-terminal fragment. And ideally sequence it by ms/ms - again likely
    > comparing it to predicted sequence.
    >
    > These can give good information about N
    > Terminii. Hard to beat old school N terminal methods for total unknowns
in
    > some cases however.
    >
    > Matt from phone
    >
    > > On Apr 21, 2020, at 2:25 PM, Yan Wang <email obscured>> wrote:
    > >
    > > I'm wondering if there's a preferred protocol for protein N-terminal
    > > sequencing using mass spec? I remember the PSRG had a study on this
    > lately
    > > but don't see a result. Any comment is more than welcome.
    > >
    > > Best,
    > > Yan
    > >
    > > ――
    > > View topic http://list.abrf.org/r/topic/4o8AxSQxvrJU3I4cluimch
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Hi Yan,

Yes, there is a protocol - it was posted on ABRF web page and should be
still there.  After PSRG study, we used the protocol on Core samples to
figure out N-terminal most region of a couple of proteins.

Here is the link:
https://pubmed.ncbi.nlm.nih.gov/27006647/?from_term=erdjument-bromage+and+pavletich&from_pos=2


If you prefer, I can also forward the pdf for your attention.
Best,

Rest of post

Hediye.

On Tue, Apr 21, 2020 at 5:25 PM Yan Wang <email obscured>> wrote:

> I'm wondering if there's a preferred protocol for protein N-terminal
> sequencing using mass spec? I remember the PSRG had a study on this lately
> but don't see a result. Any comment is more than welcome.
>
> Best,
> Yan
>
> ――
> View topic http://list.abrf.org/r/topic/4o8AxSQxvrJU3I4cluimch
> Leave group <email obscured>?Subject=Unsubscribe
>
>

For simple protein mixtures (or purified proteins), we have found that in-gel
digestion with pepsin followed by LC-MS/MS will give reproducible and
overlapping peptides with high sequence coverage (typically >90%).  This may be
worth a try.

We use same in-gel digestion procedure as with trypsin (cut gel, shrink/swell
in ambic/acetonitrile to destain, reduce/alkylate in ambic buffers) but we do
one additional shrink step in acetonitrile then for digestion we swell in 1%
TFA, 9% acetonitrile, 90% water with approximately 0.5 ug of pepsin.  Yes there
are pepsin autolysis products but with the seemingly 'random' nature of pepsin
you get a lot of complimentary and overlapping coverage.  I like to say that
pepsin digestion is not predictable, but it is quite reproducible.  Great for
determining amino acid substitutions (several peptides covering same residue),
determining the protein terminus, cleavage locations, etc.

Rest of post

Cheers

Will

-----Original Message-----
From: ABRF Discussion Forum <email obscured>] On Behalf Of Yan Wang
Sent: Wednesday, April 22, 2020 10:22 AM
To: ABRF Discussion List <email obscured>>
Subject: Re: [ABRF Discussion Forum] protein N-terminal sequencing

Thanks Matt and Joel.
In this case, I'm actually trying to quantify 3 isoforms of the same protein
starting at different M. So the only thing unique for each would be the
N-terminal peptide. Two of the 3 would lose the initial M but the 3rd one
doesn't. Trypsin, ArgC, LysC and alike won't work either as one of them have
"KKR" at 3-5 position. We are settling with GluC, which only works in solution
and is not as reliable. With that we can detect 2 out of the 3 in cell culture.
Now we are thinking if N-terminal peptide enrichment would help. I'd like to
avoid using CNBr if possible.

Yan

On Wed, Apr 22, 2020 at 12:17 AM matt sweeney <email obscured>>
wrote:

> One can use intact mass measured compared to predicted if you know
> sequence.
>
> Or one can digest the sample and then obtain the mass of the predicted
> N-terminal fragment. And ideally sequence it by ms/ms - again likely
> comparing it to predicted sequence.
>
> These can give good information about N Terminii. Hard to beat old
> school N terminal methods for total unknowns in some cases however.
>
> Matt from phone
>
> > On Apr 21, 2020, at 2:25 PM, Yan Wang <email obscured>> wrote:
> >
> > I'm wondering if there's a preferred protocol for protein
> > N-terminal sequencing using mass spec? I remember the PSRG had a
> > study on this
> lately
> > but don't see a result. Any comment is more than welcome.
> >
> > Best,
> > Yan
> >
> > ――
> > View topic
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Thanks Matt and Joel.
In this case, I'm actually trying to quantify 3 isoforms of the same
protein starting at different M. So the only thing unique for each would be
the N-terminal peptide. Two of the 3 would lose the initial M but the 3rd
one doesn't. Trypsin, ArgC, LysC and alike won't work either as one of them
have "KKR" at 3-5 position. We are settling with GluC, which only works in
solution and is not as reliable. With that we can detect 2 out of the 3 in
cell culture. Now we are thinking if N-terminal peptide enrichment would
help. I'd like to avoid using CNBr if possible.

Yan

On Wed, Apr 22, 2020 at 12:17 AM matt sweeney <email obscured>>
wrote:

Rest of post

> One can use intact mass measured compared to predicted if you know
> sequence.
>
> Or one can digest the sample and then obtain the mass of the predicted
> N-terminal fragment. And ideally sequence it by ms/ms - again likely
> comparing it to predicted sequence.
>
> These can give good information about N
> Terminii. Hard to beat old school N terminal methods for total unknowns in
> some cases however.
>
> Matt from phone
>
> > On Apr 21, 2020, at 2:25 PM, Yan Wang <email obscured>> wrote:
> >
> > I'm wondering if there's a preferred protocol for protein N-terminal
> > sequencing using mass spec? I remember the PSRG had a study on this
> lately
> > but don't see a result. Any comment is more than welcome.
> >
> > Best,
> > Yan
> >
> > ――
> > View topic http://list.abrf.org/r/topic/4o8AxSQxvrJU3I4cluimch
> > Leave group <email obscured>?Subject=Unsubscribe
> >
>
> ――
> View topic http://list.abrf.org/r/topic/7DvkYnVxfcFVjNYoC9tITD
> Leave group <email obscured>?Subject=Unsubscribe
>
>

One can use intact mass measured compared to predicted if you know sequence.

Or one can digest the sample and then obtain the mass of the predicted
N-terminal fragment. And ideally sequence it by ms/ms - again likely comparing
it to predicted sequence.

These can give good information about N
Terminii. Hard to beat old school N terminal methods for total unknowns in some
cases however.

Matt from phone

Rest of post

> On Apr 21, 2020, at 2:25 PM, Yan Wang <email obscured>> wrote:
>
> I'm wondering if there's a preferred protocol for protein N-terminal
> sequencing using mass spec? I remember the PSRG had a study on this lately
> but don't see a result. Any comment is more than welcome.
>
> Best,
> Yan
>
> ――
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Hi Yan,

Just to clarrify do you mean the identification of true protein start sites
(N-Terminomics)?

If so then the standard methodologies for N-Terminal labelling and mass spec is
the use of TAILS (terminal amine isotopic labelling of substrates).
https://www.ncbi.nlm.nih.gov/pubmed/21959240  - Protocol that our lab has
optimised for different species over the years.

The dimethyl labelling procedure of N-Termini is used +/- purification.
This paper talks about the down-falls
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631386/

Kind regards
Joel

I'm wondering if there's a preferred protocol for protein N-terminal
sequencing using mass spec? I remember the PSRG had a study on this lately
but don't see a result. Any comment is more than welcome.

Rest of post

Best,
Yan

Thank you both Yishai and Eric.

I think I will follow through on both suggestions (lab environment permitting).

I will post back the results once finished.

Rest of post

:-)

Joel

Hi Dorothy,
no idea if this helps. In a Eicosanoid project we found that after solid phase
extraction some fines remained and the backpressure went up after each
injection. It became clear to filter the eluate from the SPE 96 well plate. To
do this we put a PALL 96 well filter plate under the SPE plate and used a
vacuum manifold. Without vacuum nothing runs through the filter. Drawing our
setup would be much easier, but I try. Into the vacuum manifold, we put the 1
ml 96 well PTFE filter plate (PALL) on a normal PP 96 well plate, just in case
something runs through (never happened). On top of the manifold, we put the 96
well SPE plate (Phenomenex, AGILENT what fits the application). First we washed
the SPE and applied sample and washed - all to waste, then we eluted directly
into the filter plate. In the last step, we put the filter plate with the
eluate on top of the manifold and eluted into a 96 deep well PP plate. This got
lyophilized and dissolved in a few µl of injection solvent and directly into
the autosampler.
What I suggest, instead of centrifugation, which we tried but did not help,
filter your sample through a 0.1 or 0.2 µm filter using a 96 well manifold.
Please contact me if my description is too weird - I can look for the photos I
took
Manfred

Am 20.04.20, 19:07 schrieb "ABRF Discussion Forum im Auftrag von Dorothy R.
Ahlf Wheatcraft" <email obscured> im Auftrag von
<email obscured>>:

    Good Afternoon,

    I'm wondering if any proteomics groups can give us some advice. We're
looking to purchase a new centrifuge and ideally would like to have one that
spins 96 well plates fast enough we can load directly onto our autosamplers
rather than transferring to 1.5ml tubes, spinning to get particulate down, and
then transferring to vials. Two questions as part of that discussion came up
though, what centrifuges are good for that as most styles don't go that fast,
and if there's any issue with 96 well plates withstanding higher speeds.

    If anyone is currently using a centrifuge for this or similar and can chime
in thanks in advance!

    Dorothy
    ――
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Dorothy,

I'm sorry, but I gave you an incorrect link to the document; it's actually:

biosci-batzerlab.biology.lsu.edu/genomics/documentation/3130_Templates_EtOH_Precip_Plate-centrifuge.docx

I'm in the middle of a bunch of long-overdue updates to the website, and
grabbed the old link by mistake.

Rest of post

Scott

-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Scott W Herke
Sent: Monday, April 20, 2020 7:35 PM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] Centrifuge for proteomics samples in 96
well plate

Dorothy,

I know you were asking about centrifugation of proteins, but I this information
should still be relevant.  I use an Eppendorf 5810R for pelleting our DNA
Sanger-Sequencing reactions in an Ethanol-EDTA precipitation protocol.  For
typical use, I spin the samples at 2500 rcf for 20 min (20C).  If I want to
precipitate the very smallest of DNA fragments (i.e., ~30-35 nt) to get
sequence data within 5-10 nt of the primer, I'll centrifuge for 30 minutes.  If
I'm in a  big rush, I can reduce centrifugation time to 15 minutes, but there
is some loss of signal intensity.  The resulting pellets are so small that they
are invisible, so I think you should be able to centrifuge out your protein
particulates as well.  To remove the supernatant, I simply flip the plates
upside-down (with a foil-kimwipe cover to catch most of the ethanol) and
centrifuge for 1 full minute at 150 rcf.  Flipping the plate upside-down is a
bit scary the first time, but those pellets are really solidly stuck and I've
never had any pellet losses with our Sanger-Sequencing reactions... and, if
spun for a full minute at 150 rcf, ALL the supernatant is removed without using
any pipette tips or risking sample loss by accidentally sucking up the pellet
in a pipette tip.

If you want to see the full protocol, go to the Science Aid Center of the LSU
Genomics Facility website
(biosci-batzerlab.biology.lsu.edu/genomics/documentation/3130_EtOH_Precipitation_plate.docx).
Typically, for centrifugation, I put 96-well plates (as well as 8-tube strips)
in 96-well plastic racks just to be sure nothing happens to the bottoms of the
plate wells; however, I've done the process many times without the racks too.

Scott Herke

-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Hampton, Brian
Sent: Monday, April 20, 2020 1:23 PM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] Centrifuge for proteomics samples in 96
well plate

If you are using a polypropylene plate, it should withstand the g-forces as
long as the holder of the rotor supports the plate uniformly across the bottom.
People tend to spin particulates down in tubes with typical bench top
centrifuges capable of ~15k x g, because that works.  But sedimentation rates
have both a g-force and time component.  So if the best system you can get only
spins your plate at 6k x g for example, then you just spin for a longer time.

WRT the Eppendorf 5810, that rotor has a max speed of 4000 rpm which is ~3200 x
g.  So I would spin the plates for at least 30min to pellet particulates.  If
you have the luxury of not needing all the volume of the sample, then certainly
leave a consistent amount of volume in the wells so as to not aspirate up any
of the particulates at/near the bottom of the well.

Best,

Brian

On 4/20/20, 1:29 PM, "ABRF Discussion Forum on behalf of Ekram Hossain"
<email obscured> on behalf of <email obscured>> wrote:

    CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email
system. Whether the sender is known or not known, hover over any links before
clicking and use caution opening attachments.

    I work in a metabolomics group. The majority of our samples are Tissue and
serum. I have a ThermoFisher SPD111V speed-vac unit that I use as a centrifuge
only mode on 96 well plates to ppt out particles. When this fails, I use an
Eppendorf 5810R centrifuge. However, in my workflow I transfer the supernatant
to a new 96 well plate. So I do not have a direct comparison to what you are
trying to do. If I have to recommend, I will go with the Eppendorf one as it
has stronger rpm. I think it can go up to 14000 rpm. I am not sure about the
speed vac rpm.
    Best regardsEkram

    -----Original Message-----
    From: Dorothy R. Ahlf Wheatcraft <email obscured>>
    To: <email obscured>
    Sent: Mon, Apr 20, 2020 12:07 pm
    Subject: [ABRF Discussion Forum] Centrifuge for proteomics samples in 96
well plate

    Good Afternoon,

    I'm wondering if any proteomics groups can give us some advice. We're
looking to purchase a new centrifuge and ideally would like to have one that
spins 96 well plates fast enough we can load directly onto our autosamplers
rather than transferring to 1.5ml tubes, spinning to get particulate down, and
then transferring to vials. Two questions as part of that discussion came up
though, what centrifuges are good for that as most styles don't go that fast,
and if there's any issue with 96 well plates withstanding higher speeds.

    If anyone is currently using a centrifuge for this or similar and can chime
in thanks in advance!

    Dorothy
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Dorothy,

I know you were asking about centrifugation of proteins, but I this information
should still be relevant. I use an Eppendorf 5810R for pelleting our DNA
Sanger-Sequencing reactions in an Ethanol-EDTA precipitation protocol. For
typical use, I spin the samples at 2500 rcf for 20 min (20C). If I want to
precipitate the very smallest of DNA fragments (i.e., ~30-35 nt) to get
sequence data within 5-10 nt of the primer, I'll centrifuge for 30 minutes. If
I'm in a big rush, I can reduce centrifugation time to 15 minutes, but there
is some loss of signal intensity. The resulting pellets are so small that they
are invisible, so I think you should be able to centrifuge out your protein
particulates as well. To remove the supernatant, I simply flip the plates
upside-down (with a foil-kimwipe cover to catch most of the ethanol) and
centrifuge for 1 full minute at 150 rcf. Flipping the plate upside-down is a
bit scary the first time, but those pellets are really solidly stuck and I've
never had any pellet losses with our Sanger-Sequencing reactions... and, if
spun for a full minute at 150 rcf, ALL the supernatant is removed without using
any pipette tips or risking sample loss by accidentally sucking up the pellet
in a pipette tip.

If you want to see the full protocol, go to the Science Aid Center of the LSU
Genomics Facility website
(biosci-batzerlab.biology.lsu.edu/genomics/documentation/3130_EtOH_Precipitation_plate.docx).
Typically, for centrifugation, I put 96-well plates (as well as 8-tube strips)
in 96-well plastic racks just to be sure nothing happens to the bottoms of the
plate wells; however, I've done the process many times without the racks too.

Scott Herke

Rest of post

-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of Hampton, Brian
Sent: Monday, April 20, 2020 1:23 PM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] Centrifuge for proteomics samples in 96
well plate

If you are using a polypropylene plate, it should withstand the g-forces as
long as the holder of the rotor supports the plate uniformly across the bottom.
People tend to spin particulates down in tubes with typical bench top
centrifuges capable of ~15k x g, because that works.  But sedimentation rates
have both a g-force and time component.  So if the best system you can get only
spins your plate at 6k x g for example, then you just spin for a longer time.

WRT the Eppendorf 5810, that rotor has a max speed of 4000 rpm which is ~3200 x
g.  So I would spin the plates for at least 30min to pellet particulates.  If
you have the luxury of not needing all the volume of the sample, then certainly
leave a consistent amount of volume in the wells so as to not aspirate up any
of the particulates at/near the bottom of the well.

Best,

Brian


On 4/20/20, 1:29 PM, "ABRF Discussion Forum on behalf of Ekram Hossain"
<email obscured> on behalf of <email obscured>> wrote:

    CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email
system. Whether the sender is known or not known, hover over any links before
clicking and use caution opening attachments.



    I work in a metabolomics group. The majority of our samples are Tissue and
serum. I have a ThermoFisher SPD111V speed-vac unit that I use as a centrifuge
only mode on 96 well plates to ppt out particles. When this fails, I use an
Eppendorf 5810R centrifuge. However, in my workflow I transfer the supernatant
to a new 96 well plate. So I do not have a direct comparison to what you are
trying to do. If I have to recommend, I will go with the Eppendorf one as it
has stronger rpm. I think it can go up to 14000 rpm. I am not sure about the
speed vac rpm.
    Best regardsEkram

    -----Original Message-----
    From: Dorothy R. Ahlf Wheatcraft <email obscured>>
    To: <email obscured>
    Sent: Mon, Apr 20, 2020 12:07 pm
    Subject: [ABRF Discussion Forum] Centrifuge for proteomics samples in 96
well plate

    Good Afternoon,

    I'm wondering if any proteomics groups can give us some advice. We're
looking to purchase a new centrifuge and ideally would like to have one that
spins 96 well plates fast enough we can load directly onto our autosamplers
rather than transferring to 1.5ml tubes, spinning to get particulate down, and
then transferring to vials. Two questions as part of that discussion came up
though, what centrifuges are good for that as most styles don't go that fast,
and if there's any issue with 96 well plates withstanding higher speeds.

    If anyone is currently using a centrifuge for this or similar and can chime
in thanks in advance!

    Dorothy
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