hi

There is the possibility of protein degradation.

other possibilities :

1- non specific primary or secondary antibody binding

( sample over loading / normally 20ug)

2- kozak like position or sequence in your gene. ( produce 20 kD recombinant isoform)

you can striping & reprobing PVDF just with secondary antibody to sure non-specific banding .

good luck

Have you tried to run the samples soon after lysis? I have noticed that one of my proteins of interest start to have cleaved products with time, even when stored all the time at -20 degrees and in Laemmli buffer. 

Hi Kelly,

No I have not. I have always lysed in laemmeli then froze down in -20 C out of convenience.

Dear Sushanth,

1. If the bands are non specific, then you should be able to see the signal in your mock transfected (empty vector transfection) cells also in Western blot. If it appears only in your transfected cells then it might be the degradation product of your desired protein.

2. As Kelly already suggested, you can try not storing the lysates for a long time in -20 and then do the blot loading one lysate from -20 stored sample for comparison. 

3. I hope you add protease inhibitors to your sample before storing to avoid degradation after lysis. 

4. If you are seeing m-cherry signal under microscope, then your fusion protein is getting expressed. Try to observe it in different time points (0 hr, 18 hr, 24 hr, 36 hr, 48 hr and 72 hr) post-transfection to check the peak appearance of the signal and try to make the lysate at these different time points to check the with time appearances of the lower molecular weight products. 

5. If you have the wild type protein without the fusion tag, you can also use that to transfect and check whether similar pattern of degradation products are there or not. 

6. Also, I hope you have sequenced your cloned product to avoid any other mutations in your insert that might lead to degraded product.

Hope it helps!

Thank you Tias,

Yes I do have a negative control untransfected lane that does not show any bands. I do not add protease inhibitors I just lyse straight in laemmli. I will try lysing in a lysis buffer in the future and storing the samples in lysis buffer until blotting. I have sequenced the cloned product and it looks fine.

Hi,

I once had the same problem with transfecting mCherry-fusionprotein in Hek293 cells: I found the fusion protein, pure mCherry and a degradation form in between.

For me lysis buffer worked way better than laemmli. In addition I noticed, that "real lysis" with lysis buffer and ultrasonic cell disruption lowered the amount of degradation product further. Maybe you can split after resuspending the cells in your lysis buffer and compare both conditions. Hope sonication helps you, too.

Dear Sushanth,

I think you might want to consider adding protease inhibitors to your lysate. To check expression of my construct, I generally lyse in RIPA buffer and add protease inhibitor cocktail and then store in -20. After quantifying the amount of protein then I load the required amount in gel for western blot. If even after adding protease inhibitors you are getting those lower molecular weight bands, at least you'll know whether your protein is getting degraded after lysis or it is unstable and therefore getting degraded inside the cell itself. 

Hi Christiane,

That's very helpful! May I ask what lysis buffer you used?

I wouldn't be too concerned Sushanth. I'm transfecting 293T cells with mEGFP, mCherry, mCitrine, and mKOK fusion proteins and am seeing degradation products for each of the fusion proteins when analyzed by Western blot (even when protease inhibitors are included).

In my opinion, it results from overexpression of the protein and is a common occurrence. The fragments are likely proteosome-processed protein that is being turned over. Completely normal. If you express them in another cell type or with a weaker promoter you will likely see the intensity of the bands decrease.

Hi Sushanth,

I used different lysis buffers, because I really wanted to get rid of those bands. I mainly changed the tensids, e.g. NP-40, Deoxycholinsäure, Triton, Tween... and their amounts, but that did not change anything. In the end I used normal RIPA with addition of 1 tablet/10 mL buffer "cOmplete™ ULTRA Tablets, Mini, EDTA-free, EASYpack Protease Inhibitor Cocktail" from Roche. I discarded the left-overs from the buffer after every experiment, because the inhibitors didn't like freeze-thawing in my hands. If you aliquot, that might be different.

From my experience with that specific expression, I agree with Erik. You probably need to chance the whole system (promotor, cell line,...) if you want to get rid of the degradation band completely. Maybe transfection with less DNA also helps to decrease the overexpression rate and therefor degradation.

You could try treating cells with MG132 proteasome inhibitor to see if degradation is reduced. Be aware when you are doing fluorescence microscopy that if proteasomal degradation is occurring it means at least some fraction of your protein is being misfolded and likely does not localize correctly in the cell. Also, I believe there have been reports that some proteins are sensitive to boiling with SDS, with SDS-dependent cleavage occurring at specific sites. Depending on the protein of interest, we normally treat samples at anywhere from 37° to 95° prior to successful PAGE/blotting (for other reasons, mainly intractable aggregation of large membrane proteins). Boiling is seldom necessary and is known to cause problems with certain proteins. 

Generally, I would aim at isolation procedure in this case.

Looks you have good experience with fast protein isolation;) but it seems too ..unbuttoned;) If you use proper lysis buffer (RIPA works), low temperature at all times and inhibitors then you can wonder on more complicated questions.

Also, you mentioned that after thawing your isolated protein samples you boil it (100deg also seems hazardous;) i'm using 95-98) spin down the aggregates and run it - are those aliquotes or are you re-freezing the previously thawn sample? Anyway, refreezing's bad, yeah obviously;), and storage in -20 isnt the answer for times longer than 1 month, unfortunately.

Best!