Da (Daltons), kDa (kiloDaltons), MWCO (Molecular Weight Cut Off) & estimating protein size from

The molecular weight (MW) of a compound tells you how much 1 mole (6.02 x 1023 copies) of a molecule weighs. It’s given in g/mol (or Daltons (Da), where 1 Da=1 g/mol) 
When dealing with biological macromolecules like proteins, MW is often given in Daltons (Da) or kiloDaltons (kDa) 
 
1 Da = 1 g/moL 
1 kDa = 1000 Da = 1000 g/moL 
 
you can think of the Da as shorthand so you don’t have to write out g/moL 
 
more here: blog: bit.ly/molecul... RUclips: • Working with molecular...  
 
quick back-of-the-envelope estimates:  
 
the average amino acid is ~0.1 kDa so a 1000 amino-acid-long protein would have a MW of ~100 kDa 
 
the average DNA nucleotide is ~300 Da, so multiply length of double-stranded in bp by 300*2=600 to get a rough idea of MW 
If you see a MW for a protein or a MWCO listed that’s in thousands, you’re probably dealing with Da not kDa. Unless you’re dealing with something super massive!
 
In addition to knowing where in relation to your molecular weight ladder you should look for your protein on a gel, some of the main times you really need to think about the MW is when dealing with dialysis membranes or spin concentrators (ultrafiltration devices)....
“Centrifugal ultrafiltration” which is just a fancy-dancey way of saying you stick your too-watery protein solution into a membrane-lined tube insert and spin it really fast. The force from the spinning pulls the water (plus salts and other small things) through the membrane, but your protein’s too big to get through the membrane’s pores so it stays put. Sounds pretty boring - and it is - especially when your protein is taking hours to concentrate to the desired concentration… but it’s really important and we do it a lot so today’s a practical, post I hope will bore you not…Some details on the what and the how and then some of the why’s (preparing for SEC, preparing to freeze, buffer exchange, etc.)
blog form: bit.ly/spincon...
Protein concentrators come in many volume-holding-capactities (e.g. 0.5mL, 4mL, 15mL) & molecular weight cut-offs (MWCO) (e.g. 3K, 5K, 10K, 50K). MWCO refers (indirectly) to the size of the membrane’s pores. It’s given in units of Daltons (Da) & tells you molecules below this size can go through (are penetrating) but molecules above this size are retained (are non-penetrating & stay in the top). You want to choose a MWCO smaller than your protein (& anything else you want to keep) but larger than whatever you want to get rid of.
You put your sample in the top chamber & spin it in the centrifuge.
Molecules smaller than the MWCO are pulled through the membrane into the lower (waste) chamber, but molecules bigger than the MWCO stay in the upper chamber
The bigger the pore size, the faster you’ll reach equilibrium (because if a molecule bumps into the membrane it’s more likely to “bump into” an open space it can get through & doesn’t have to worry as much about “squeezing” through. BUT you want to be careful not to select a size too close to your protein size since the MWCO is an average, so you still might have pores big enough to let your protein through.
Typically, a MWCO “guarantees” that at least 90% of molecules of that size will be retained. BUT proteins have different shapes which MW doesn’t account for (e.g. a long skinny protein might be able to “slither through.” So to avoid losing protein, you typically choose a MWCO 1/2 the size of smallest thing you want to keep.
Note: this is the same situation you face with dialysis
Another important thing to keep in mind is that, since it’s an average pore size and since all the proteins are still able to mix around with one another, it’s NOT useful for separating proteins by size. Ultrafiltration can only be used to separate things that differ by a magnitude of size. So I can separate my protein from salts, but not from another protein.
Also, since we’re on the topic of salts, you can use this as a way to “desalt” a protein and/or switch it into a different buffer - concentrate the protein and then re-dilute it in the buffer you want.
I usually concentrate it in spurts of 15min or so depending on how much concentrating I need to do. In between spurts I use a pipet to mix around the liquid, especially near the membrane, where gunk can build up on the membrane walls and make passage more difficult.
Especially if you're dealing with a protein you haven't worked with before, be on the lookout for any signs of it crashing out (precipitating). That can happen if you try to concentrate things too far, but how far "too far" is depends on the protein! (And the salt concentration, etc.)
Finished in comments

[@thebumblingbiochemist]

There are a couple of times during the protein purification process when you want/need to concentrate your protein
1. before Size Exclusion Chromatography (SEC) (aka Gel Filtration)
2. before freezing your final product
for details: bit.ly/spinconcentrators
here’s a link to download today’s cheat sheet: drive.google.com/file/d/1TqCRh23TO8s5T23DjBcJeCUWl23ps9sI/view?usp=sharing  
 
and here’s a concentrations cheat sheet: drive.google.com/file/d/1v7pff7cEpJTvr-IADLwdwwFOlEDdz8Ny/view?usp=sharing   
 
more about concentrations: bit.ly/sciencestocksolutions ; RUclips: ruclips.net/video/RjXYvOiLh2w/видео.html & ruclips.net/video/wC623-AGBOs/видео.html 
 
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com                            
                              
#scicomm #biochemistry #molecularbiology #biology #sciencelife #science #realtimechem

[@christianwalterskirchen4331]

Very helpful! Keep up the good work 😄

[@thebumblingbiochemist]

Great to hear!

[@user-ec1so9hq2t]

When calculating moles of a polymer (or protein), do we just divide the grams of a polymer by its molecular weight? I came across a research paper where they utilized hyaluronic acid (80 kDa), and they said that 1g of hyaluronic acid to be 2.492mmol, but if I calculate by dividing 1g by 80,000g/mol, I get 0.0125mmol. But I figured out that the calculation becomes correct if I divide 1g by hyaluronic acid's monomer's molecular weight. So I'm confused whether which way of calculation is correct.

[@thebumblingbiochemist]

Things get tricky with reporting molarity of homopolymers, especially those with varying lengths - both ways are "right" but one gives you molarity of the polymer and the other of the monomer. So it depends what you're interested in finding