Hi,
One of my colleagues has used a pre-cast, commercially available 10% separating gel for his SDS PAGE. Later, while interpreting the results, we observed a broadening of the marker lane towards the bottom of the gel (image enclosed). Some claim that he has faultily documented an inverted gel image while we are sure that it was done appropriately. On the internet, we found people have documented similar broadening of the marker lane (image enclosed). However, it would be nice if someone can support the same fact.
Hi, @navnit and all others.
I think you should check your molecular marker because some companies prepare them not be heated anymore. In this case, my personal opinion is: as soon as you got it from the company, do prepare aliquots and maintain them all at the right temp.
About the Image, the one on the left is not of the same dimensions as yours, in my opinion, what makes difficult to be sure about similarities . .. are they the same marker maker or not? Did you check the Gel size (mini tanks or midi ones)???
Lastly, your picture (right side) shoes only 7 bands, the other one (left side) has 10 bands. On the upper Image of yours, it seems to present the blue ending line of stainer (coomassie brilliant blue R) there. If it is correct, your Gel is upside down.
My recommendation would be to do a new gel and mark it by cutting the corner of your gel to give you orientation positioning to know the up and down places. Secondly, check the time needed to keep all bands of your ranging marker in the Gel.
Wishing you all the best.
Hope it helps.
I supposed you used marker Pierce™ Unstained Protein MW (26610) Markerhttps://www.thermofisher.com/order/catalog/product/26610?SID=srch-hj-26610
But photo from internet containes info about another marker Bio Rad http://www.bio-rad.com/en-es/sku/1610363-precision-plus-protein-unstained-protein-standards-strep-tagged-recombinant-1-ml?ID=1610363
Dear Antonina,
Yeah, you are right about the marker we have used. So, does that cause a difference in the way the bands order themselves (widening towards the bottom). In general, I want to know if these gel images are right or are they inverted as some people claim?
Navnit Kumar Ramamoorthy if you work with commercially available 10% separating gel (shelf life is ok) you have some problem with sample preparation of marker ( 10 minutes at 95 °C to completely denature the proteins ), electrophoresis buffer or reduce the load per well ( recommended - Mini-gel: 5 µL per well (0.75-1.0 mm thick) or 10 µL per well (1.5 mm thick)
I used Pierce™ Unstained Protein MW (26610) and obtained clear not-broadening bands. Of cours, your is gel is normal for visualization but you can check reasones of broadening
Range of 10% PAGE SDS is about 25-250 kDa
If you have doubt where is up and down, cut a small piece of gel in the right and corner and you will know the orientation ( or use Multicolor Range Protein Ladder)
Thanks Antonina, I believe the gel is normal as you have mentioned too. I needed a clarification since some people have been calling it an upside down image. As for the resolution and clarity, we were able to figure out the required recombinant protein at the designated MW range.
I agree with younits not a upside down image. I am not an exoert on this, but on my little experience, i would just like to remind you to be mindful of rhe correct orientatiin of the gel, correct voltage, appropriate time and temperature for heating the sample, appropriate load per well, correct compisition of the running buffer, remembering to remove the tape on the bottom, run time not exceeding 35-40 mins. I am sorry if you ensured all of these and i could not provide any useful information.
Dear Navid,
Thanks for your honest opinion and your expert advise.
Hi, @navnit and all others.
I think you should check your molecular marker because some companies prepare them not be heated anymore. In this case, my personal opinion is: as soon as you got it from the company, do prepare aliquots and maintain them all at the right temp.
About the Image, the one on the left is not of the same dimensions as yours, in my opinion, what makes difficult to be sure about similarities . .. are they the same marker maker or not? Did you check the Gel size (mini tanks or midi ones)???
Lastly, your picture (right side) shoes only 7 bands, the other one (left side) has 10 bands. On the upper Image of yours, it seems to present the blue ending line of stainer (coomassie brilliant blue R) there. If it is correct, your Gel is upside down.
My recommendation would be to do a new gel and mark it by cutting the corner of your gel to give you orientation positioning to know the up and down places. Secondly, check the time needed to keep all bands of your ranging marker in the Gel.
Wishing you all the best.
Hope it helps.
Good point, the marker inused does not need to be heated as well like the samples.
Dear Adriano,
Sir, we used the marker as per the manufacturer's instruction. The gel, which we did later, had no issues and conveyed details appropriately. There was lot of skepticism surrounding this particular gel; Hence, I wanted to get expert opinion/suggestion. Thank you for your suggestions, sir.
HI, Navnit Kumar Ramamoorthy
You're more than welcome. That's what researchgate is all about. Helping others.
Anyway, can you tell us what is your final results about it?
All the best.
Yes sir,
We got the recombinant protein expressed at a MW, somewhere close to 50 KDa. The gel, which we had cast after this one had proper alignment. Thank you so much for your advise, sir. :)
HI, Navnit Kumar Ramamoorthy
Great to know!!! Congrats and good vibes for you and your work ...
Why don't you just leave the wells and stacker attached to the resolving gel?
No doubts then!
Hello
Yes , the samples and the unused marker does not need to be heated.
Good Luck
Broadening of bands during electrophoresis in the separating gel occurs by diffusion, small proteins are more affected than large. To reduce this, use a gradient gel: A molecule ahead of the pack will be slowed down, trailing proteins will be accelerated by the different %T in their environment.
Generally keep electrophoresis experiments as cool as possible, diffusion is temperature dependent. A cold room serves more to chill the operator than the equipment, use the cooling loop of your equipment instead.
Glycoproteins have broad bands In SDS-PAGE because the oligosaccharide contains variable numbers of negative charges. Use CTAB-PAGE instead to counteract this effect (doi:10.1016/S0003-2697(02)00639-5).
Dear Prof. Engelbert,
Thank you for the detailed information and advise. It is very educative.
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