HI,

Have you used the standards before and did it work?

Did you try using another blocking solution? say BSA.

This may not be necessary but I am curious. Did you check your transfer conditions? May be the concentration of the approx 90kDa band is high so some part of it may have transferred.  Is your protein of interest is transferred as well?

You can write the manufacturer/dealer about the problem. May be it is an issue with the lot so you can either get a refund or another batch of standards. 

I have been using fermentas pre-coloured molecular wt standards. Works fine without any problems unless the transfer is messed up. 

Hope it helps.

Santosh

Hi.

It´s the first time that I have used these standards. I have not used another blocking solution, it could be it...

I don´t think is the transfer, since my protein, around 72kDa, is there. My transfer conditions are 15V, 30 min, in a semi-dry transfer.

I wrote to the manufacter and still waiting for a response...

Thanks for the suggestions. I´ll check with the BSA

Hello Nancy,

I am curious to know whether u have used the protein markers for checking the size of your protein of interest or for other purposes during electrophoresis. In first case, the blocking solution wont have any affect on the detection of protein markers, as they can be seen by naked eye on gel and membrane before and after transfer of proteins. But, yes, the marker provided to you by company may not meet the qualities.

Secondly, if you are probing the standard with the antibody for doing some of your experiments, then, yes, blocking by BSA and skimmed differs a lot. Milk contains many proteins mostly phosphoproteins, and acts a strong blocking agent than the BSA. But in cases where one would be probing phospho proteins, the use of milk is not appropriate as this will results in high background on the blot while probing with phospho antibodies. The use of fatless milk in blocking is due to the reason of hyrophobicity of the fats, as they hinder between the interaction of antibodies and proteins, and between pvdf membrane and blocking agent (which are hydrophillic). So, if you get a weaker band/signals during immuno detection, you can increase the amount of protein to be probed, or you can use BSA upto 5% instead of skimmed milk to block your membranes, but the other possible conditions can never be over ruled.

Lastly, if you are doing these experiments using NATIVE-PAGE, you cannot use the prestained protein markers (which usually have SDS) for size determination, may be the protein is not being seperated/ diffused, in that case you be able to it is tricky to immunodetect them.

I hope it is helpful

Saleem

Hello Nancy,

To save your problem, frist doing the non -SDS gel electrophoresis with out using standard protein , to known howmany bands  your sample have. Second,it is better to you using BSA  or casein as a strandard protein which they are molecular weight a round 67 KD and 23KD. I hope it is helpful.

Essam

Hi Saleem,

I use the SDS-PAGE, so is probably the blocking agent...

Hi Essam,

Thanks for the suggestion.

Hola Nancy, It, is strange thah you only se a big protein because the smaller ones transfer faster than biger.Anyway you can use any prestained marker that you can see the resolution without stain before transfer. We use 150 volts 1 hour for minigels of 1.5 mm and to know how the transfer has gone, stain the gel after the transference. I think that your transfer conditions are quite low, increase the voltage and time. Buena suerte

no I have not used the standards