Maria Benitez-Guijarro 22:32
We have two DC powders, one that I made last year (BUT it had matrigel on it, so we will probably have matrigel contamination if we use this powder...) and the powder that I sent from Spain this year, 3 g of powder (from 45 big plates) growth without matrigel - So from the best powder, we only have 3g
lacava 22:33
Yes, use the 3g that is the best material - but, with the matrigel, my assumption is, it will spin out during centrifugation - does it spin out?
If it spins out - we can use that stuff still, I think
So, maybe use the BOTH, and we just see what happens - better to use the material than not to, IMO - as long as we can be sure the gel doesnt certainly ruin it
If it forms a pellet it is fine - I guess we are just not sure the mass of the gell?
and that is the main issue?
Maria Benitez-Guijarro 22:36
It pellet, but not totally
lacava 22:40
I think that, with the matrigel one we just accept it, and use it if we can estimate the difference
I think that the only problem matrigel poses is that we dont know how much cells we are actually weighing…
Have we done an IP and looked on an SDS gel - if you did a 50mg IP or something, side by side with same amount of beads, you could run both on SDS-PAGE and western and compare the ORF1 signal
if they are very simialr, then the gel is just not a problem
The MS wont even see it when we search, because we dont search mouse proteins
The instrument would see it if the peptides are present, but the software wont match it to any proteins… so, as long as it is not super abundant, it’s fine
I think doing a small scale test IP is wise, and anyway makes sense before using 3-6 G
Right?
Maria Benitez-Guijarro 22:55
Oh I see...
The thing is, with 3g of the good powder, We only have enought for 3 replicates of anti-ORF1 and 3 of control, or 4 of anti-ORF1 and 2 of control...
lacava 23:03
So, this is my point -
use the matrigel powder as a low risk way to do a pre-check
Make sense?
Maria Benitez-Guijarro 23:03
When you mean pre-check, you mean doing only a couple of replicates instead of the big experiment?
lacava 23:04
I mean doing an IP western and general protein stain - if you have a little bit of PCs left, you could do the same - you want to know - is the ORF1 yeild comparabkle?
OK, so, then - let’s plan the tester experiment then…
lacava 23:06
I would say you need a DC vs PC anti-ORF1 comparison by western and coomasie or sypro
lacava 23:06
We want to see two things: (1) comparable ORF1 recovery and (2) the gel lane from the matrigel doesnt look CRAZY
Did we ever do any exoperiment with the matrigel powder?
Maria Benitez-Guijarro 23:07
Last year - But we never looked at it again, because we thought that the band we were detecting in Wes was not ORF1
lacava 23:08
OK, so, we only did Wes?
Maria Benitez-Guijarro 23:08
And gel
lacava 23:09
Looks like no problem
Maria Benitez-Guijarro 23:09
DC seems to have less ORF1 than PC
But still detectable
lacava 23:09
So, can we get the ORF1 recovery higher by using more beads or more powder?
In the gel, we dont really know what band is ORF1
lacava 23:10
We just know that on the Wes that band is ORF1, right?
Maria Benitez-Guijarro 23:10
Considering that this DC had the matrigel, the good powder should have more ORF1 for the same concentratiomn
lacava 23:10
Yes, I agree
But I wish we could figure out the % of matrigel
Do you know how many plates you needed before to get 3g copared to now?
We could estimate
Maria Benitez-Guijarro 23:52
For the matrigel BBs, I seeded 12 dishes, and got 2 g
Without matrigel, I seeded 45 and got 3g (lets consider that 15 of the dishes were at around 70-80 % of confluency when I harvested them - I was testing different cell dilutions, bc some of them could detatch without matrigel and I had to harvest at that confluency bc otherwise the cells would die) - so lets say 3g without matrigel from 40 plates
It seems that... the matrigel doubles the weight
lacava 00:11
I like it
So, let’s also assume that binding TIME is not limiting and we KNOW binding capacity is not limiting
We simply DOUBLE the amount of powder when using the matrigel material
yes?
Maria Benitez-Guijarro 00:12
Yes
lacava 00:12
So, we can do an experiment :)
Maria Benitez-Guijarro 00:12
But then, which size should that experiment be
What scale
lacava 00:13
Well, first you just need to use enough to get signal on western and stained gel - 100mg I guess
Maria Benitez-Guijarro 00:13
If we have 2 g of powder from the matrigel powder...
For PC powder I needed the equivalent to 25 mg to detect ORF1 (Western)
lacava 00:14
The purpose of this experiment is simply to determine how much DCs powder is needed to get a PC like result
Maria Benitez-Guijarro 00:14
Yes
lacava 00:14
So, you could do 25mg PC vs 50mg and 100mg DC?
Maria Benitez-Guijarro 00:15
Yes - matrigel DC or both DC?
lacava 00:15
Matrigel DC only
we are going to assume is has 1/2 the ORF1
Maria Benitez-Guijarro 00:15
Maybe 25 mg of DC would be as 25 of PC...
And 25 is not much
lacava 00:16
My understanding is, when we ran this experiment before with equal amounts, there was less ORF1 in DC - so me it looks like much less than 1/2
If you are SURE to get ORF1p signal from 50mg of PC, then set that as the baseline
Maria Benitez-Guijarro 00:17
Totally sure
lacava 00:17
Then you need to find how much (matrigel) DC gives equal ORF1 - we would use 1/2 that much non-matrigel DC in the real experiment
Yeah, then you could save material and use 25
Once we see how much DC gives ~the same ORF1 as PC, we can organize the real DC experiment better