Good morning all,
I've been having issues for the past few weeks with my Western blots. I have done several westerns in the past years, without ever seeing a gel with this specific appearance.
This is what I have done so far: I have done a trial run before running my samples, as none of the instruments had been used in a while and I had just remade all the stock solutions. The trial run looked a bit better than this, but still thick. However, actin on that membrane came out looking fine, so I proceeded with samples.
The run went horribly, looked uneven, all samples were spotty and the ponceau looked full of "holes". I have tried and made several more gels, changing the solutions one at a time (trisHCl, APS, acrilammide, SDS). None of it makes any difference, so I don't know what could have possibly gone differently from the first run to the one with my actual samples (the marker ran unevenly too, so it's not the samples).
I have both tried a run with the running buffer from the first trial and with a newly made one, they both look like the picture. Temperature is not high, when I ran my samples I quantified protein content for each well and they had 20ug, I tried loading four different sample buffers, this is the result. Any idea what I could try next?
In the picture here you see a well loaded with 10 ul of 5x sample buffer, 30 ul of my sample (20ug of proteins in it), then 30 ul of 2x sample buffer.
Thank you to anyone who might offer insight!
Hi Giorgia Bresciani
Can you elaborate more details such as:
From where did you extracted your samples? and what is the extraction buffer?
How long the stacking gel is?
What are the running volt, ampere and time?
Hello Mohamed Khashan , thank you for taking the time to reply. The proteins are extracted from ARPE-19 cells and I used a RIPA buffer. However, I have run into the same issue with my markers as well, it runs as if something were "obstructing" the run. I usually start the run at 60V for half a hour or however long it takes the samples to run the stacking, then I switch to 100-110V for the rest of the resolving gel (around one hour and a half usually). I usually pour the stacking to be around the same depth of the wells or slightly smaller. I'd say around 7 to 10 mm.
Giorgia Bresciani it looks like you have a problem in your gel and the mostly in the stacking one. We are using the same system (Bio-Rad). so I will suggest you the gel recipe we are using hope this will give you a hand. Pls give an extra attention to the PH of Tris buffer used in the preparation in the stacking, separating gels and running buffer.
1. Stacking gel: 212 ul dH2O, 1 M Tris HCL (PH=6.8) 380 ul, 40% acrylamide 225 ul, 2% APS 150 ul, 1% SDS 30 ul, TEMED 4 ul ... Total volume (3 ml).
3. For a 10% seoarating gel: 2250 ul dH2O, 1 M Tris HCL (PH=8.8) 3750 ul, 40% acrylamide 2500 ul, 2% APS 500 ul, 1% SDS 1000 ul, TEMED 10 ul ... Total volume (3 ml).
The PH of the running buffer should be between 8.3 to 8.8.
Make sure to put your gel in the room temperature for at least 40 min before starting.
Leave the gel for one day after preparation in +4.
Make sure to wash the wells well before loading your samples.
The stacking gel should be around 1 cm under the comb.
Hope that would help and looking for good news.
Uneven running of a western blot can be caused by a number of factors, such as: 1. Incorrect gel concentration or gel thickness. 2. Poor protein migration due to incorrect sample buffer composition or an insufficient amount of sample buffer. 3. Poor transfer of proteins from the gel to the membrane due to incorrect transfer buffer composition, incorrect transfer time, or incorrect transfer voltage. 4. Poor blocking of the membrane due to incorrect blocking buffer composition or an insufficient amount of blocking buffer. 5. Poor antibody binding due to incorrect antibody concentration, incorrect antibody dilution buffer, or incomplete antibody incubation.
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