I have a problem with unequal loading of protein in WB (my housekeeping gene varies considerably across conditions).
Here's what I do:
Cell lysis with RIPA buffer and inhibitors for 30 min on ice. Spin down 10 min at 10k x g and collect supernatant. Measure protein concentration (BCA) and mix together 20 ug sample, 3x loading buffer, and 5% beta-m; boil, load into gel.
My question is whether the unequal volumes could be responsible for unequal loading of my housekeeping gene, even if each lane gets the same total amount of protein? But without adding a diluent to all but the least concentrated sample, the volumes are different in each well. Do people usually equalize the volumes in WB? And with what - lysis buffer, dH2O, or 1x loading buffer?
Another question I have is about storage and measuring protein concentration. I noticed that a lot of protocols say that after lysis you can measure protein concentration and then freeze. But I've been snap freezing my lysates and when I'm ready to run the gel I will thaw the lysates, spin them down, and do the BCA. Is there a reason not to do this?
I have other problems not limited to unequal loading, so if anything looks wrong please let me know.
If I'm going to use the protein for western, I usually process the samples as quickly as possible to get it to a boiled, denatured state. Then I'll freeze them and not worry about degradation. If for some reason I cannot process them, I'll freeze them in the presence of protease inhibitor, then thaw, measure BCA and boil/denature. Basically, I make sure the BCA is done immediately before boiling. I never measure, then store, then thaw, then boil.
As for the change in the volumes: I usually normalize concentration of entire batches of biological related samples (all samples from a single run on a single experiment). So, all samples will be at 1 ug/uL, or 0.5 ug/uL, etc etc. So when I run a gel I'll load the same volume for all samples.
Hi,
I normally load equal volumes in everything but the ladder. To get it all up to equal volumes, I use in-house dH2O. The water comes from our building, so I have no idea what kind of quality it is. Better than tap, at least. That works fine for me. I have, in the past, loaded unequal volumes. I didn't see a difference at the time.
I like your method better. I store my lysates at -80C and I'm paranoid about weird things that can happen during a freeze-thaw. I think it's best to do your protein assay and then load right after. Or, at most, store the lysates at 4C. I use water as my diluent for the BCA.
Here's a few weird questions that have come up in my work though!
How are you revealing it? Are you making sure that your HRP is evenly spread and mixed? And is it not expired/bad lot?
If you use x-ray film with an automatic developer, is the developer working well? If someone doesn't maintain it, algae grows in the tanks, rollers gouge film, the heater wires malfunction, and all kind of annoyances.
Is your BCA assay compatible with your lysis buffer? I don't use RIPA, but I use a DC assay instead because I have more detergent.
Hi Joe,
I agree with Dr. Jordan R. Yaron for the process samples as quickly as possible to get denatured form by mixing with loading buffer, boiled and storage.
I will also normalize the loading volume of each samples by deionized water and make sure the volume of loading dye in each samples is equal.
Best,
Joe, I normalize my proteins with lysis buffer containing 1X protease inhibitor (I keep all solutions the same throughout) before addition of the denaturing Laemmli solution. I do my best to keep all the properties of the solutions the same (ion content, etc) to avoid gels running at odd angles (smiling, frowning, etc).
If your concentration of sample is the same, your lanes will all be loaded with the same volume. If your samples all have the same protein, salt, etc concentration, your gels will run beautifully.
That makes sense - perhaps I'm getting some artefacts from different salt concentrations, etc.
Weber - is there an advantage to storing samples after adding loading buffer and boiling rather than storing them right after lysis? After boiling them can you store them in -20 or even 4 degrees? for my experiments, it would be difficult to do the lysis, protein quantification, concentration adjustment, adding loading buffer, and boiling at the same time.
Colin - now that you mention it, perhaps my HRP secondaries are too old. I'm having a hard time getting the bands to show up without really concentrating the primary or secondary antibodies. At the moment I'm using new primaries that are validated for WB. We develop using ECL, so I don't think the development part is the issue. Maybe we need new secondaries.
Joe, if necessary you can add protease inhibitor and store in -80C with minimal loss in protein integrity. -20C might be okay for a day or two, but I would absolutely avoid keeping unboiled proteins even with protease inhibitor at 4C for too long.
I agree with Jordan. I like to run my samples for western blots right after I perform cell lysis. If western blot can be perform after harvesting cells, I wash cells with PBS and store the cell pellet in -80C. Bringing all the samples at same protein concentration is a good idea. You can always normalize your band with respect to housekeeping protein using ImageJ.
Hi,
I totally agree with Jordan. I do I prep my samples that can be either fresh or being stored at -80 C (then in liquid nitrogen) as quick as possible. During that, the preped samples that are lysates now in ice. Then, centrifuge them and take the supernata and I usually separated each sample on multiple aliquates to make sure no thawing and freezing cycle might affect the protein content. I measure the amount of the protein and then normalize the samples accordingly, dd the sample buffer, boil, and lastly (if you are not proceeding with western at the moment) save in -80C. With that I make sure not to worry about thawing and my samples give good western results.
Good luck!!
If the values are too much variant I would suggest do a blot with housekeeping genes. And then normalize using the band density .. and then load accordingly . u should get them equal and ur test protein levels can be quantified without doubts.
Hello Joe,
I read a lot of nice answers regarding the best way to store your samples, but I have a different question for you.
In your protocol do you wash your cells with PBS or NaCl solution to remove the media before you collect your cell pellets for lysis?
If you don't wash, then you have a lot of protein background due to the media FBS and this can explain the variability of your housekeeping proteins.
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