Could you kindly help me to specify what could be possible cause of my problem with band in Western blot as showed in my attach picture? picture 1 is 40 kda, picture 2 is 60 kda.
1. Some time, the band is not straight, a bit deformed like the leftmost band in the figure 1.
2. I often get the band which has faint or disappearing in the middle part as most band in both figures and especially second rightmost band in the figure 1.
I use Biorad's Mini-Protean system, 10well, 1.5cm comb, sample solution is 5 uL/well, homemade SDS-PAGE gel: 12% resolving and 4% stacking, SDS running buffer, running at 60V for 10min then 200V for 39min, fresh Towbin with 20% methanol transfer buffer, clear bubbles with glass stick, transferring at 100V for 1hr. The membrane is PVDF, wet with 100%methanol for 1min and transfer buffer for another 15min.
Thank you so much
Cheerio,
The concentration of protein seems to be very less. Try to increase your protein concentration. There may be bubbles left while transferring.
It seems to be gel-pushing sample enloading with the tips. There are a sort of tips for PAAG protein samples with elongated like proboses ending, they are more suitable for vertical gels recommended also for clear 2D.
We had similar problems some time ago and it helped to just prepare all the buffers freshly. Maybe precipitation or evaporation changed the salt concentrations and this resulted in 'ugly' blots.
I always measured pH in diluted stocks before casting PAAG, sometimes new buffers should be prepared. Poor protein solubility is also possible. Then dample rehydration in lower salts buffer is useful.
Hi, did you control the transfer with ponceau? If the ponceau was good, the problem colud be in the further steps. Pay attention to incubation step, the blocking buffer and the antibody used. The uneven/ patchy aspect could be caused by antibodies binding to the blocking agent or secondary antibody aggregated. Use fresh blocking and antibodies or filter the used antibodies if are expensive.
Hi, your bands are different from dumbbell bands, because you have an uneven signal instead in the dumbell you have the signal but it is distorted in a smile effect. I would recommend to do ponceau to the membrane to see if the problem is at transfer level (for example you have to add more 3MM sheet paper, to make more strict the sandwich), at blocking level, at incubation (antibodies or ecl); try to isolate the "incriminate" step.
Hi, in order to solve a "crooked band" problem, start loading your samples not from the very edge (although sometimes it is impossible if you need to use all 10 wells). Thus, in your figure 1, you should have additional, sample-free wells loaded with the loading buffer only (with/without the size marker) at each side of your series of samples. Also important that you make sure that all your samples have an equal amount of the loading buffer.
Regarding the second problem, to me it looks like there is an unequal amount of the substrate used on the middle area of the gel. The background looks different over there. Alternatively, as it has been noted by Elena Yu. Filinova, you may have jus loaded smaller amount of sample due to the accidental gel pushing.
If the bottom of your wells in the stacking gel are uneven (maybe bubbles when placing the comb) the protein may also run unevenly giving you wavy bands in the end, like in your first figure.
Thank you Valeri, Arunas, and Magdalena.
For the egde well (leftmost and rightmost) I do use for the ladder and prestained standard.
Elena and Arunas, May I ask what you mean by "gel-pushing"? The volume is too small? or the load is too heavy to push gel?
Yes as Magdalena pointed out. I do sometime have some bubbles between comb teeth and gel. There is no bubble when I pour stacking gel fully but the bubbles appear after I place comb. Beside Degassing the gel (I don't have any equipment to degas the gel), is there a way to prevent this kind of bubbles?
For me, it looks like transferring problem. If the sandwich is not tight it leads to unequal transferring of protein and of course increasing the amount of protein will also help.
Had this problem as well. Looks like either transfer problem (proteins transferred incompletely) or solubilization problem (not fully solubilized proteins shed epitopes). Try adding DTT to your samples or indeed, making a tighter sandwich
All the possibilities above are good but work through this methodically.
Theses bands are tight so the stacking gel is working well and this implies your buffers are all good.
The twist could be poor polymerisation of the resolving gel. Uneven distribution within the band could be loading or western bloting techniques.
1. Resolving gel - make sure it is well mixed by swirling without creating bubble but acrylamide solutions must be degassed, as suggested, before adding the TEMED and APS.
2. The uneven loading within bands could be:
a) badly shaped wells, make sure the upper well is full of running buffer before pulling out the comb (stops wells collapsing as it immediately rushes in as you pull the comb out slowly).
b) Poorly dissolved sample - Sample in loading buffer - heat in a boiling water bath before loading
c) As everyone else has suggested poor transfer as bubbles in the way of the bands when western blotting. Take particular care to push away bubbles in that region of the transfer.
Good luck and do report back.
The causes of that can be multiple, but definitely you cannot make a relevant conclusion from quantification of non homogenous bands.
It can be affected by the amount of protein you load and the buffer (salt concentration or other). in my opinion the quantity you use 10ug/well is perfect (or 20ug if your protein is low expressed in your cell) , I don't think the blots you show has bubble problem. however you did not mention the solution in which you extract is. avoid high concentration of salt (>200mM), always load same volume and solution in every well to allow nice straight migration.
I would not go that high in voltage. I advice to use fixed current (about 20-22 mA/gel of miniprotean size) with max voltage fixed at 125V. In this situation, at the beginning of migration, 22mA/gel should give you around 70 Volt, and it will increased with time and stop increasing when it reaches the fixed voltage (125V). In this condition the migration quality will be nice and more reproducible from time to time.
make sure your glass are well clean
I am not familiar with PVDF transfer so I cannot give you advices for this step. but keep always everything clean, and wash your gel for few seconds in transfer buffer to remove some acryl residues that could affect a nice transfer to the membrane.
I find that gently washing the wells with stacking gel buffer (transfer bulb pipet works well) prior to loading well disolved protein removes unpolymerized acrylamide that may push protein to the sides of the well, especially if your gel loading tip is always in the center of the well. Also I have observed that if you let the tip touch or puncture the stacking gel near the middle, it tends to have the unexpected effect of pushing protein loading to the sides of the well, contributing to the uneven load - so I try to never touch the stacking gel during loading. Rapid loading also tends to swirl the protein being loaded out to the sides and then it eddies up the walls of the well, remaining more lateral than even in distribution. You can observe and see if that is occurring. I also agree that removing any visible bubbles is key to quantifiable results. Also with PVDF two-step wetting, I find that equilibration in running buffer for several minutes (gentle agitation) really cuts down on release of any trapped air during the run - so fewer bubbles form. Much of this can be controlled and the few minutes of extra time during a load is well worth even, quantifiable bands.
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