What do you mean by 2nd dimension not run at all? 

Hello Nazilah,

Yes, as suggested by Biswapriya, faulty quantification may be responsible for a false impression of high protein concentration. I will always suggest not to rely on Bradford assay. BCA is pretty good for secretome protein quantification.

Can you tell from how many cells or flasks did you extracted your secretome and how did you concentrate and processed it? Then only we can have a guess on false protein conc.

in case the amount is low, you can do silver staining and in 2D it is recommended to do silver stain as you won't want to miss any spots.

You are welcome for further suggestions

Hope it helps..!!

hi Kumar,

I use BCA for protein quantification.

Basically, i culture the cells with completed medium (10% FBS) and the initial number was 1 miilon cells. After the cells confluece (80-90%), i wash the cells and incubate with serum free for another 48h. Finally i collect  the media ,centrifuge for 10 minutes to separate the debris and continue with concentrate the media using ultra amicon centeifuge,10kDA (Milipore).

The final volume of concentrate media was around 200-250uL. Then i proceed with BCA.For this sample, i've got 68 ug/uL.

For dehydration step,  i used 400ug sample + sample buffer(300uL).

Are there any part i could improve others than silver staining.Should i increased the protein concentration?

Hi Nazillah,

There could be many reasons for this result:

- Loading to the 1st dimension doesn't work. So, you should try different loading procedures (passive vs active vs cup loading).

- Loading to the strip is ok, but focusing is not ok. Check your buffer containing sample: if there are high salt concentration, SDS or other detergent contamination? For this, you also can see during focusing that voltage can not reach high. If this is the case, removing salt or detergent contaminants by dialysis or aceton precipitation is necessary.

- Focusing is ok but proteins did not move from strip to gel. Stain your strip after running the 2nd dimension to control if all protein move to the gel or not. If this is the case, change 2nd dimension condition: percentage of gel, voltage, sealing between strip and gel.

- If all above are not the case, stain your gel with more sensitive agent (Sliver staining, Sypro ruby). 

Kind regards,

I would suggest you include a QC step after you complete the protein assay. Run a 1D gel on 10ug of your protein and stain with coommassie. If you can see good protein bands, then you know that your protein quantification is okay.

The next step would be to make sure that the IEF separation has actually occurred. One of my students had a similar problem - the electrodes were not properly connected, but the Powerpack showed the normal increase in voltage and current as if all was well. She did not check the duye front and proceeded to the second dimension. There were no spots at all as the diffused protein was lost from the strip during the equilibration steps. You may choose to run an extra IEF gel that you can stain with coommassie - you should see well separated bands as well.

HI Stephen,

I did run 1D gel (SDS) in order to check my protein. Yes, i've got a band on 1Dgel. However, when i run 2D, there was no spot obtain.Actually, i also guest there is something problem at IEF stage.

From your response above, am I correct in understanding that you had a single band in the 1D gel? If this is so then there is nothing wrong with your 2D gel as i can see what appears to be protein spots of the same molecular weight just below the 5th molecular weight marker. This would imply that your secreted proteins are very low in abundance and you must have a single serum protein dominating the secretome fraction!

Nazilah, there are many possible reasons you're not seeing any spots, and it could be difficult to trouble shoot without more information regarding your methodology at all steps.

When you remove complete media you should wash the cells repeatedly with PBS (or some other physiologically relevant buffer) prior to addition of the serum free media. You indicated that you washed the cells, but how often? I've washed cells 10X and still see a substantial presence of albumin and other high abundance serum proteins, so if you only wash 1X a tremendous amount of these proteins remains and they will contribute a very large proportion to your total protein concentration.

As Stephen indicated, it's always a good idea to run a 1D gel after protein quantitation. If you have a scanned image of the 1D gel it would help with trouble shooting as it would give a general idea of your sample complexity and relative abundance of various components. For instance, you mention that you have a band on the 1D gel, but 1 band doesn't sound like a complex secretome sample to me. Also, your estimated protein concentration of 68 ug/uL seems rather high, as I typically get 7-7.5 ug/uL following Amicon concentration (40 ng/uL unconcentrated, so 180-200 fold concentration), as measured by Bradford assay, and I have no problem using Bradford, by the way. The differences between our concentrations could depend on the type of cells you're using, culture conditions, how much you washed the cells, and the fact that I collect conditioned media after 24 h instead of 48 like you did, but a 1D gel will also give you an idea of whether your BCA was accurate or not.

When the IEF strip is run with sufficient protein you typically see opaque bands where the more abundant proteins focus in the IEF strip. They're not always highly visible, but if you examine the strip from various angles there is a difference in light refraction that generally allows you to see whether bands are present or not.

If you're still seeing nothing after the 2nd dimension, it also doesn't hurt to retain the IEF strip and stain that after running SDS-PAGE to see whether protein remains in the strip or not. If protein is there, that could indicate rehydration issues, especially considering the 2nd dimension ran okay based on your marker lane. If you see nothing in the IEF strip and nothing in the 2nd dimension gel, it's possible that protein is not getting into the IEF strip in the first place. What IEF conditions are you using? Passive, active, cup loading, etc. (as mentioned by Nguyen)?

There are various troubleshooting techniques to examine each step in the procedure, but this should at least help with getting a sense of where your protein is and how complex it is. It's even possible that you're not getting protein into the strips at all. Attached is an old 2D gel of mine run on a Protean-IEF system (70 ug protein, silver stained pH 3-10, 15% SDS-PAGE, not great resolution in the basic region unfortunately) to give you an idea of what you could be seeing with silver staining. Also wouldn't hurt to silver stain your 1D gel to check complexity if it is dominated by 1 or 2 high abundance proteins.

Hope this helps, and good luck with your gels.

Looking at your gel again, it appears the broken section in the lower right is not oriented correctly. The upper right should be on the lower left, so that the bluish stain runs up the right side of your gel. The part that goes up the right side looks like your ion front that should be along the bottom of your gel. If you flip it around, I think you'll also find that it aligns better with the curvature there in the main section of the gel.

Hi Kent,

Here is my 1D picture. The one in red box is the sample i've used for 2D (as shown in previous picture). So, it showed i have no problem with my protein sample since right. I guess there might be a problem during IEF or dehydration. currenty i am repeating again and will see the result  soon.

Thank you for the suggestion.