It will normalize for the amounts loaded equal volume of samples , we normally ponceau stain our membrane and image it before blocking and use the bands to show equal loading.

Ponceau staining of your membrane before blocking is the easiest way. It is good to monitor sample loading, and protein transfer, but not good to monitor antibody incubation and signal detection.

Hi Rosa,

I can tell you two things that I used to do. First of all, I would measure the amount of the total protein per supernatant. Then I would adjust the volume to obtain the same total protein concentration per sample. Further I used to do a house keeping protein such as GAPDH or beta-actine in my westernblot as a standard to measure each band digitally and compare their relative expression levels.

Good luck,

Ali

For western it would be better to quantify total proteins using  biochemical assays like BCA before loading.  once transfer is done, you can see your total protein bands using ponceau staining or thermofisher has new blue dye for staining proteins on the membrane, before adding antibody.

Thanks, I usually look my Ponceau but although I seed the same number of cells, I put the same ml of culture and collect the same volume of supernatant cell culture, sometimes the ponceau shows inequal loading, so I repeat the experiment. 

In the culture media you have FCS and that have a BSA. So in the supernatant of the culture you have a BSA that you can detect by an antibody directed against it. If that works it will be the best control for your loading.

I hope that helps 

Before loading proteins into a gel, I always perform a BCA or Bradford assay to ensure that equal concentrations of protein are being loaded. Then I confirm with a membrane stain.

If you don't care that the amount of proteins are unequal, then I would load several known concentrations of BSA from a standard curve and compare my unknown samples to the curve. Membrane staining alone is not always indicative of protein concentration.

you can consider panseau staining as Ramachandran said. Abderrahmane Bengrine suggestion to take BSA as a standard is quite acceptable(Since BSA is present in supernatant i.e.,10% FBS we use, and concentration can not be altered easily after long term incubation with cells).

Thanks, my cultures are in arresting overnight and are treated without serum, so I dont think BSA is a good idea.

Dear Rosa,

you may use BSA even if you cells are without serum for over night just add a known quantity of media with serum to your sample and load all in the gel. If you worry about diluting your simple you may use protein precipitation before loading the samples.

Reagards

Quantify first the amount of protein in the supernatant

Unfortunately, you can't. There are no proteins that are constitutively secreted from cells in such a stable manner that you can reliably use it to normalize blots, as you might with Na/K-ATPase, Actin, Tubulin or GAPDH. An option is to generate a stable cell line that DOES secrete a protein constitutively and to use those cells for your other experiments, but then you have to ask whether that extra burden you are placing on your cells is affecting their behavior.

For secreted proteins a common method for normalizing blots is to run both supernatants AND cell lysates from the same population, and to use your normalized protein from the lysates to show that you are detecting secreted proteins from approximately the same number of cells.

This is, of course, imperfect. For example, what if variability was introduced during the supernatant preparation? This is where honesty and stringency come into play. You have to be very, very careful to make your supernatant samples consistent and handled equally.

You could, feasibly, do a BCA/Bradford on the supernatant samples, but you run into the possibility of there being other factors secreted, of which you are not aware, that change depending on your conditions. Also, if your medium contains any serum your values will be hidden in the background noise of the albumin.

I see the lysate/supernatant blots very often.

The current discusion does not tacke the question of  what do you want to normalise for ? and what do you wish to compare ?

Increased production of something PER cell ?, then you have to normalise against cell mass, so you have to use a cellular protein (if you do macrophages HLA  or the usual tubulin etc) , , equal seeding of cells is only apparent, you still might get different cell numbers after you experiment.

I you wish to compare differential amounts of proteins in your supernatant by W blot, then albumin woudl have been a good call (good suggestion , also fibronectin from the serum woudl have been good, as macrophages do not make much themselves ! ), however, in the absence of it you will have no benchmark comparison from "outside" of the cell.

I would advise against equalising protein content, as you want to see differences of secretion, right. What if a couple of proteins are made in really large amounts ? If you then equalise per protein content per well you might have "equalised" away the true secretion differences. I woudl warmly suggest to refer back to cell mass/numbers - not as seeded, but as measured. So take the supernatant for your W blot, concentrate, i necessary, and keep the cell pellete for DNA analyses (depends how sensitive) or run a Wblot for some stable macrophage protein as representative of cell mass. Equalising for cell lysates in terms of protein content is OK because you could expect a dominance of more stable proteins. But equaliseing for secreted proteins could lead you in a trap. Best wishes Michael

Ponceau works well but you have to be careful, if you are using the licor then you will get some background. But on nitrocellulose it shows really good bands. 

well, the best an only beside keeping the volume and dishes the same (adhesion of proteins)

I never tried but, why cant we add known amount of BSA to the Serum free medium and use that medium to treat cells and collect after incubation time then go for concentration procedures. This can help to maintain equal amount of protein after concentration(rather than relying on pipetting) by measuring the intensity of BSA band after panseau / commassie staining of gel.

Thanks, I will treat very carfully my cultures and the same my supernatants, in parallele I check the same in cells.

we also struggle with similar concepts. particularly when trying to publish. Reviewer's often ask to show some normalization factor. We were doing this on a volume/cell basis. Therefore, one could collect all the cells at the end of teh experiment, and by total protein or total DNA prove that the same number of cells per condition, in fixed volume are used. We have typically perfomed experiments in always 2mL of serum free media with 250,000 cells, precipitated total protein in conditioned medium, then lysed all cells for either DNA count or total protein quant.

I think there are a few options, none of them are perfect. (i) The most simple thing to do is to measure total protein of your lysates and load the gel with equivalent amounts of protein. (ii) you can sacrifice one of your replicates and measure viable cells either by viability staining or by visually or instrumental counting and load the gels with equal number of cells (iii) stain the blots with a second antibody directed against a stable marker present in your culture and normalize on density measurements.

My preference is simply to assay and normalise the protein concentration after sample preparation, run 2 gels per blot - one for blot transfer, one for Coomassie blue stain, then simply use densitometry of the Coomassie stain as a loading control, as per "Coomassie Staining as Loading Control in Western Blot Analysis - Charlotte Welinder and Lars Ekblad".

It's not perfect, but not at all bad for a quick and dirty method.

You can go for MMPs as control, they are zymogens and are secreted, MMP2 and 9 are so far the best matrix metalloproteinases to go for, mainly because these are perhaps the best characterized of the family with lot of comercial antibodies well validated.

You should also be able to detect actin in conditionned media, not because is secreted but because there will always be cell lysis and it is a pretty abundant cinstitutive protein. For sure it is not a secreted protein but I think it could be a sort of semicuantitative help to determine how many protein IS in the media.

Are you using centrifuge concentrators to concetrate your conditionned media?

hope these points help

JP

Thanks to answer, and yes I concentrate my 1 ml of supernatant culture media (without serum) by acetone precipitation. 

Hi,

We have the same issue in our lab, we use TCA/acetone to precipitate the proteins.

I normalise the ECP(extra cellular protein) with ICP (Intracellular protein)

Step 1; Note the volume of lysate for each well

Setp 2; Determine the ICP concentration using BCA assay or any other.

Step 3; Now you have the volume and amount of protein per well, just normalise this to ECP.

TADA, Trust me it works most of the times.

Hello, 

thanks, I will try,

Rosa

Dear Friends,

I want to analyze the secretory factors (Interleukins/Cytokines/Chemokines) using ELISA and LUMINEX based assays in conditioned medium. Unfortunately, at the time when supernatant was collected from treated and untreated samples, the number of cells were significantly different between samples and I missed to count the total number of cells in each sample. Is there anyway that I can quantify the total protein concentration from cell culture supernatant, in order to normalize my data??

Very impressive question and discussion, I don not think that anybody will concentrate equal volume of concentrated protein even with the concentrator available. It's my personal experience, even volume of water can not be concentrated equally in available concentrate.  Staining method is also a good option, but secretion of protein is not equal all the times, small factors (endogenous and exogenous) can change the secretion. 

My suggestion for this is that, use the ready-made gels available, which without any staining showing the image of total protein loaded in your gels and use this image as a normalizing factor during the normalization of your blots. an application available in chemifluorescence instruments. Hope this will solve your issue of loading control.   

Hi, we had the same problem with supernatants of cultured endothelial cells.  In our hands you can normalize the actin content in the supernatant to total protein in the supernatant or other target proteins. I am not quite sure if this works with your macrophages. Otherwise you can count your macrophages and normalize the detected protein amount of your target gene in the supernatant against the number of cells. Or you normalize against the total protein amount or DNA content of your cultured cells after cell lysis. Good luck!