I use 1x ttbs, wash it 4x for 5 mins and 3x for five mins after transfer, and before blocking. I use everyblot from biorad as the blocking agent and for antibody incubation. Thanks!
Hi Camilla,
something went very wrong in this analysis. I even suppose that there was a combination of several unfavorable circumstances.
The biggest problem is lanes 2-4. These samples are clearly overloaded. Verify the protein concentration of this samples. Moreover, it is likely that due to the placement of the electrode, part of the sample was "pulled up", resulting in visible streaks on the membrane. A possible reason for such an image is also an error in the preparation of the gel for electrophoresis (incomplete polymerization).
The ladder is pale, indicating excessive rinsing that led to bleaching of the bands. By the way, werify the quality and specificity of your antibodies. Check if they are suitable for your target protein and have been properly validated. Using high-quality antibodies from reliable sources is crucial for obtaining clear and specific signals in Western blotting.
It is possible that in this case there was an incomplete transfer of proteins from the gel to the membrane. Insufficient transfer can result in weak or missing bands on the membrane. Next time ensure that the transfer of proteins from the gel to the membrane is efficient and complete. Confirm that your transfer conditions (time, voltage, buffer composition and volume) are appropriate for the size and nature of your proteins.
The background of the membrane is very gray (possibly the result of the photo), indicating that it has been stained for too long. I understand that this was due to pale striations, and I probably would have done so myself, too, but after drying the membrane it distorts the image even more.
Unfortunately, the analysis is correctable once the above errors are ruled out. Chin up, next time will be better.
Good luck
Hi Jakub, thanks for your insight! I'll re-doing the blot and will post here the updated version. I just want to ask you a couple of questions regarding your tips:
1. When you talk about the placement of electrodes, what exactly do you mean? That I switch the anode for the cathode? If that's what you are asking, no, I put the red with red and black with black... or do you mean that the lid wasn't placed properly?
2. I use 8ul/lade of the protein dual color ladder from bio-rad, how much do you usually use? Also, do you rinse/wash your membrane after transfer and before blocking?
3. Unfortunately, the protein of interest does not have a whole lot of commercially available antibodies for the specific epitope I'm investigating, and this is actually the best antibody candidate - which still required a whole lot of dilution optimization to be detected - hence, I'm trying to load the max amount of protein possible per lane. I'll try to go down a knot to avoid the smeared bands! Thanks particularly for that tip!
Hi Camila,
I agree with Jakub in terms of overloading. The protein sample from one lane seems to overflow into another lane. I use the BioRad precast gel, and I usually load less amount of protein in each lane than its capacity. For example, if the capacity of the lane is 50ul, I usually load 40ul or less.
Another issue that may cause this problem is the fat content in the protein sample. Before you load the protein sample, you should remove all the fat(if present).
Best of luck with your experiment!
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