Can you share the image of the blots? How does ponceau staining look?

1. How much total protein are you loading? Please check protein extract with commassie staining to ensure it looks like a total protein extract- if the extract is ok then

2. after transfer please stain the membrane with Ponceau S (reversible water soluble stain) to ensure that protein transfer is even. if ok then

3. if you are using film, then the Xray cassette is defective

4. if you are detecting chemiluminesence using an instrument like Typhoon or chemidoc, make sure that the exposure is not too long- this will cause loss of signal.

Yes, please put an image of the blot.

It can be due to protein degradation over time or poor sample preparation. Make sure your proteinases are up-to-date and working. Use a combination of proteinases.

For working with proteins the lab should be cold- around 17 degree centigrade is good. You should put everything on ice to make sure your proteins do not degrade.

Regards

It is not possible that beta-actin totally disappear. But this actin isoform is not always the best marker to quantify proportionally the other bands since its quantity could sometimes vary depending on the differentiation state of the cells or of the cell type.

Are the proteins you are looking for of a far higher MW than actin ? or at the opposite have they a lower MW and thus implies a short transfer time ?

Sometimes, bands are totally missing due to a poor transfer quality in this part of the gel/nitrocellulose paper. Did you check the quality of transfer by Ponceau red staining and did you see all the presumed actin bands ? Another alternative is to run parallely 2 gels loaded with the same protein samples, one for staining with Coomacie blue and one for the transfer. Then, after the transfer you can also stain with Coomacie the transfered gel to evaluate the quality of transfer. The blue gel can also be used as "loading control" to compare the immunostained bands from the second gel.

Good luck for your experiments.

It could also be simply you are stripping the protein from the membrane, during your membrane stripping protocol. Maybe reassess your protocol, or as recommended previously, check the membrane with ponceau?

Or alternatively, beta actin is not the most reliable loading control for your cell type. There are total protein stains you can use and image to use in your analysis, or a variety of other potential loading controls.

Based on your target protein, it maybe more suitable to probe for your loading control following your initial proving, without stripping the membrane. There are some excellent guides to loading controls if you google it.

Hope you find a solution, good luck.

hey guys, I tried everything you said, now the targets all work, but the beta actin does not.

I increased the loading to 50 yg- still doesn't work. I tried to immediately stain the actin, without stripping before.

The samples were thawed on ice and homogenized in porcelain bead tubes with ice cold RIPA buffer with proteinase inhibitor, spinned down at 4°C for 20 mins (15.000 g x av) and kept on ice the whole time. I use image fusion for the fotos, so it will expose the membrane for 1 sec and later on suggest an adequate exposition time- it works for all the others. Maybe somehow the actin really degraded. But why do the other targets still work?

Hey Nadia,

do you know if the antibody works well? Maybe it's a problem with the actin-AB

Going off of Katharina Bomans said, I'd suggest ditching the beta-actin antibody loading control- you could try probing for a different protein (e.g. GAPDH), or do a ponceau s stain of the whole membrane instead (which is what I would do). Staining the membrane with ponceau s would allow you to look at total protein expression, rather than just beta-actin, so that should solve the issue, regardless of what the actual cause is (antibody, b-actin expression, etc.).