I would like to ask a question regarding protein extraction and Western blot. I seeded cells on amniotic membrane scaffold and proceed with protein extraction on day 1, 7, 10 and 14.
The problem is after electrophoresis and transfer there were big blobs of protein in each well. It doesn't happen when I load protein which I cultured in T75 flask. It only happen to protein I cultured on amniotic membrane.
Is the serum from culture media cause the big blob?
Protein extraction method: Amniotic membrane seeded with cells was subjected to liquid nitrogen and was crushed with mortar in pastel and collected to eppendorf tube. It was incubated for 20 minutes with 500ul Nacalai Tesque Ripa with protease inhibitor. Sample was alliquote to 100ul each and stored in PCR tubes at -80'C. Protein concentration was quantified using Bradford Assay and most of the time I will get an average of 2ug/ul of protein in each sample.
For Western blot 40ug protein was mixed with 4x Laemmli plus mercaptoethanol and incubated at 95C for 5 minutes (updated) and was loaded to 10% gel and electrophoresized at 100V for 90 minutes. Protein was transferred to PVDF membrane activated with methanol for 60 minutes (updated 60 seconds).
Membrane was stained with ponceau and I attached the picture for reference. The second picture was taken after protein was imaged using a digital imager.
Does anyone know why I have big blob around 50-75 kDa and the protein seems stuck there and don't want to move down beyond 40kDa?
Some proteins samples also doesn't have good amount of Beta Actin (second picture). An indication of protein degradation of it just don't have enough protein?
In the picture the most right is protein ladder, then some samples of protein seeded on amniotic membrane and the most left lane was protein from cells seeded in T75 flask.
Does it happened because my protein samples degrade? I need to visualize 12 protein of interest and your opinions will be much appreciated.
Thanks!
The blob is likely caused by air bubbles between your gel and your membrane when assembling the western blot sandwich. The little white spots that you see on the thick bands are also caused by either air bubbles or trapped padding remnants between your sandwich.
I haven't used the Nacalai Tesque Ripa with protease inhibitor buffer (I usually use an in lab recipe), but looking on the company's website it says that this buffer lacks SDS. Are you performing a native or denatured western blot procedure?
From the procedure you described, I'm not sure if the samples were prepared properly for denatured western blotting. Ideally, you have to add SDS, glycerol, bromophenol blue and B-mercaptoethanol (similar Laemmli buffer recipes are online) and boil your samples before loading your gel.
How big is your protein that you are trying to detect? You might want to tweak your blotting time/voltage depending on the size.
Also, while b-actin is widely used as a reference gene, it might not be the best option for your samples. Depending on the conditions your cells were exposed to, b-actin might actually show differential expression. Try other reference genes such as GAPDH. You can also normalize your analysis against the coomassie stained membrane. A lot of articles started to use this method, you might want to take a look into this way or normalization.
Best of luck,
Thanks Mirada and Rashad for your recommendations. I would like to clarify that I made a typo when writing about activating PVDF membrane in methanol. It was 60 seconds actually.
I also heat samples in 4 x Laemmli buffer with mercaptoethanol for 5 minutes at 95C. The ratio of sample to Laemmli buffer 3:1.
I also roll the membrane and the filter paper when preparing the sandwich transfer. Therefore I am quite sure that the blob is not air. But I maybe wrong.
I tried to find proteins around 66 KDa which is exactly in the blob area.
Hi, have you found an answer to the big blob? I have the exact same issue with media protein. I see that there's indeed some air bubbles during transfer but i dont think that's the cause of the blob of band that doesn't go down.
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