1. Several factors could contribute to the discrepancy between the expected band size and the observed band size, including incorrect antibody specificity, improper antibody concentration, or reduced protein expression or degradation. You may need to re-examine the conditions used in your experiment and compare them to the manufacturer's instructions, or try using a different antibody.

2. The presence of a strange shape in the middle of the membrane could indicate a blocking issue, however other issues such as the presence of non-specific binding, improper washing, or poor transfer of protein to the membrane could also be contributing factors. To determine the cause, you may need to try adjusting the blocking conditions, optimizing the washing steps, or re-examine the transfer conditions.

1. I would double check how you calculated your band size. Did you include the tag? For example if I have HA tag on my protein I would need to include the weight of the tag in addition to the weight of HA.

2. I think this looks like a bubble from when you transferred your proteins from the gel to the membrane. If you use dry transfer, make sure you are rolling out the bubbles.

Overall, I would recommend including a positive control.

For example, if your protein has an HA tag use a cell lysate of cells expressing an expression plasmid for a protein tagged with HA.

dear Andrew Kim

The problem seen in the photo is because there was a bubble between the SDS page gel and your nitrocellulose paper or PVDF, and when there is a bubble, all the proteins accumulate on the outer edges of the bubble and They are transferred to the paper and when you put an antibody on the paper, instead of creating a lighter background, it shows the sum of all the background in those edges densely. In short, you see the background accumulation of the bubbled area at the edges of the bubble.

Regarding the shift of your band from the expected place to another place, you should know that sometimes proteins undergo band shift depending on the amino acid composition. I have faced such a problem several times. I recommend that if this protein has already been expressed and described by another researcher, read its articles to see how the position where your target protein should be placed in the gel is reported (don't just rely on the position predicted by the software).

Regarding the unsuccessful target protein transfer on to the paper, I strongly recommend that you run a positive control protein that reacts with your antibody to ensure the health of the antibody and the other conditions for the correct transfer of the target protein to the paper.

Hi Andrew Kim, when there is little protein when developing, the membrane has to be exposed for a longer time and this can lead to the appearance of stains on your gel, so I recommend increasing the amount of material applied on the SDS-PAGE. The bubble that appears in the center of your figure could be air between the membrane and the gel, so when assembling the blot, be careful not to leave any bubbles.

The membrane above shows three distinct things. One is that the proteins were not transferred, secondly assembly of the gel, sponge and filter paper may not have been properly done to ensure absence of bubbles if you used a blot module as explained by Hannah. Third the membrane seems not to be properly washed. I would recommend you optimize your transfer step adequately, increase the washing time of your membrane after exposure to primary and secondary antibodies and ensure your primary antibody works optimally. The dilution ratio of some antibodies recommended in the packing list or data sheet is not always cast in iron.

dear Victor Ikumawoyi

If the proteins were not transferred to the membrane, the protein marker should not be observed on the membrane.

It seems that all the proteins with the same size in the protein marker must be bound to the membrane.

@Masoud Really? My experience with Western blots shows that if your transfer process is seamless, you'd see expressed bands on your membrane on imaging though they may be unspecific binding.

dear Victor Ikumawoyi

The prestianed protein ladder consists of proteins stained with dyes that are covalently attached to these high purity proteins. As a result, if the protein transfer process to the membrane has a problem, the proteins ladder will not be transferred to the membrane. In the past, when pre-stained protein ladders were not so common, "Ponceau S" staining was used to ensure the transfer of proteins to the membrane.

Thank you everyone for the answers! I am in the process of troubleshooting my western blot! Wish me luck!

Andrew Kim Goodluck!

I'm sorry to hear that you're having some issues with your Western blot experiment. Here are some potential reasons for the problems you're experiencing:

To troubleshoot these issues, you could try the following:

This blotting clearly shows that the washing wasn't done properly.

I will add a little of what Ahmad Al Khraisat sir has already explained, answering to the first question, yah it might have binding to the alternate splice varient protein, or sometimes protein gets hydrolyzed due to proteases and shows the band at the low molecular weight. So, I recommend checking for the Protease/Phosphatase inhibitors you are using.

answering the second question, there is a problem with washing, kindly check your TBST buffer quality. the possible reason for getting that space within the band is , while transfering to the membrane ,there could be bubble on the gel which hinders the transfer . By observing the blot you develop i can say the substrate and luminol solution got oxidized.

these are my observations, correct me if i am wrong.

Thank you everyone for the recommendations!!

Andrew

It is possible that proteases or phosphatases are contributing to the low molecular weight band observed in the Western blot. Using protease and/or phosphatase inhibitors during sample preparation and handling can help mitigate this issue.

Regarding the space within the band, poor washing of the membrane or poor quality of the TBST buffer can indeed lead to residual background signal and interfere with the clarity of the band. Additionally, the presence of bubbles during the transfer process can indeed lead to incomplete transfer of the protein, which can result in gaps or spaces within the band. Finally, the oxidation of the substrate and luminol solution can also contribute to poor signal quality and reduced clarity of the band.

Ahmad Al Khraisat could it be that the absence of anti-oxidants could be the cause? The buffer we use for SDS-PAGE (MOPS buffer + anti-oxidants) usually contains 2% of anti-oxidant but we forgot to order it so we have been using the above buffer without anti-oxidant and it has worked for some of the WB experiments... but could that be the reason? But what about the WB experiments that worked? is it dependent on sample? Like for some samples, MOPS w/o anti-oxidants work but some don't?

My apologies, I am still in the learning phase of these things...

Andrew

There are several factors that could be causing issues with your Western Blot. Here are some possible explanations for the problems you described:

• Protein degradation: The integrin subunit may have degraded during sample preparation or storage, resulting in a smaller than expected band size.
• Post-translational modifications: The integrin subunit may undergo post-translational modifications (e.g. glycosylation) that affect its migration on the gel.
• Gel conditions: The gel conditions (e.g. percentage of acrylamide, pH, etc.) could affect the migration of the integrin subunit, resulting in a different band size than expected.

• Incomplete transfer: If the protein did not transfer properly from the gel to the membrane, it could result in a weird shape in the middle of the membrane. Make sure that the transfer conditions are optimized (e.g. voltage, time, buffer composition, etc.).
• Non-specific binding: The weird shape could be due to non-specific binding of the antibody to the membrane. Make sure that you are using appropriate blocking conditions and that the primary and secondary antibodies are specific to the protein of interest.
• Contamination: The weird shape could be due to contamination of the membrane with some other substance. Make sure to handle the membrane with clean gloves and avoid touching it with anything else.

In terms of troubleshooting, you could try optimizing the transfer conditions (if you suspect incomplete transfer), adjusting the blocking conditions (if you suspect non-specific binding), and making sure that your samples are properly prepared and stored to avoid protein degradation.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw