equal loading on western blot?

[kstovall]

Hi everyone,
I need to load equal amounts of protein on all lanes of my gel - I have been performing a Bradford and just calculating the amount to load (so each lane has a different amount of sample - but the same amount of protein). Is this ok? Or do I need to adjust my samples so that they have the same amount of protein and then load the same amount of sample (like 10ul?). If this makes any sense...

[Allison M]

I would think that it'd be preferable to adjust each sample so you are loading the same mass AND the same volume of protein in each lane. For one thing, assuming your loading buffer has reductant (usu. BME) in it, you'd have the same ratio of protein to reductant in each lane. I can't back this up with any data, but it seems prudent, and a minimal amount of work.

[blilly]

I am new to bichem methods. can someone explain how much SDS should be used for cell pellet.
I am using 100ul of 2x sds sample buffer 900+100ul DTT. load 30ul, but i dont get even bands on GAPDH.
is there a 1x recipe.

blilly

[blilly]

I am new to bichem methods. can someone explain how much SDS should be used for cell pellet.
I am using 100ul of 2x sds sample buffer 900+100ul DTT. load 30ul, but i dont get even bands on GAPDH.
is there a 1x recipe.

blilly

[Anastasia_S]

here is what I do:
collect samples, store at -80
on the day of SDS-PAGE, I defrost the aliquotes for protein assay and do the assay (note-all diff conc.)
then i do my calculations on how much volume of each sample I need to give me a bit more protein than I'm going to load in a well (say I load 15ug, so I take volume tat gives me 20)
then I add ice cold (from freezer) acetone at approx x10 the volume, spin at 13000rpm for 10 min at 4C (this is to precipitate protein, and separate some of the crap that dissolves in acetone)
the I aspirate or pour off the acetone and let prot. pellets dry (if you leave acetone, stuff will float and not sink into the well)
than add my cracker buffer (1% SDS and other stuff including B-mercaptoethanol and bromophenol blue) and resuspend pellet. Amount added will depend on the amount of protein in the pellet, so that I have between 0.5 and 1 ug prot per uL cracker buffer.
Then I denature proteins at 95C for 3 min
And load into the wells. Now, I have prepared more solution more than I'm actually loading so that I don't have to scrape every drop out of the tube to make it even loading. This way I get the same amount of protein and same volume into each well.
I think if you have too much protein per uL SDS, it may not coat the protein properly hence running time will be wrong. Having said that, I had a few mistakes when I miscalculated and had x50 the amount of protein per uL of cracker buffer and although the gel looked totally overloaded, all bands were in the correct places.

Hope this helps

[Allison M]

Now, I have prepared more solution more than I'm actually loading so that I don't have to scrape every drop out of the tube to make it even loading. This way I get the same amount of protein and same volume into each well.

I do this too, and it also gives me extra for a do-over if needed...

I've recently noticed that gel loading tips are not as reliable as regular tips (p10 in this case) for loading equal volumes, has anyone else noticed this? I think the super thin tip causes significant variation due to capillary action. Grr argh.

[blilly]

how do i calculate the amount of lamelli for samples

Thanks

[Anastasia_S]

I noticed that the thin tips sometimes give variable loading, but I haven't connected it with the tip itself, thought it was my pipetting! A very good point, thanks, Allison M!