Hi,

Just a couple of questions that *might* give some clues -

- Do you use PVDF or Nitrocellulose? - I've heard that high grade PVDF membrane can bind more protein and proteins are less likely to diffuse through it.

- What percentage gel do you use, do you use a gradient gel?Transfer conditions, no. of 'volt-hours'?

- Do you visualise by AP, using ECL on film or using fluorescent secondaries?

- How do you standardise the amount of protein sample that you load so that you are convinced that you'll have enough to identify by Western blot?

I reckon there's lots of potential possibilities so apologies for bombarding you with loads of questions :)

Hi.

I was having lot of problem with this protocol. I used the kit "Protein Subcellular Fractionation Kit for Tissues" from Thermo Scientific for protein extraction and worked well. Solved my problem.

I too had experience a problem in the transfer of Histones.

Try reducing the the voltage and keep it for more time. (I generally use 30v for 70 min) .

Hope you have run 15% gel.

Try incubating the secondary antibody for 3hrs.

All the best... :-)

My student encountered the same problem, which in the end was solved by doing an acid extraction of histones, described in his article, which appears in the current issue of Genes and Cancer (Di Paola et al., DOI: 10.1177/1947601912457026)

hi.. please see the antibody is conformation or sequence specific. Since, you are getting result sometime and sometime the band is missing............this may be a sign of conformation specificity of the antibody. So, please check the catalouge of antibody. Rest i think that the other suggestion to be followed.

Thanks for all the comments and suggestions.

I have been using either a 12 or 15% gel and transferring to a 0.2 um nitrocellulose membrane. The company I purchased the antibody from has verified that it works for Drosophila, so it is likely that it will work for my fly.

I am going to try the 70 min transfer at 30V and incubating with the secondary antibody for 3 h. If that doesn't work, I will probably try the acid extraction.

Thanks again!

In 12% gel you'll never get histones...it will run out of gel.

Use 4-20% gradient gel. Or use 15% or 18% gel and stop run before the dye front reaches the leading edge of gel.

Histone is one of the protein with high abundance unless it is a variant histone. Nitrocellulose membrane is OK it binds histones without a problem.

Use the recommended concentration of antibody (by the manufacturer) in 5%milk or 3% BSA . I would recommend 5% milk with 0.01% sodium azide. Use primary antibody overnight at 4 degree C or cold room in a shaker.

Histones are small proteins..so the transfer time 1hr at 100V should be more than sufficient.

Never use sodium azide for secondary antibody. Secondary antibody should be 1hr at room temperature.

Use ECL developing.

Hey Julie,

To detect histones I prepared the extracts according to the protocol of abcam (http://www.abcam.com/ps/pdf/protocols/Histone%20extraction%20protocol.pdf). Abcam also provides a very detailed protocol for histone western blotting that worked out nicely in my hands (http://www.abcam.com/index.html?pageconfig=resource&rid=28&pid=5).

http://www.abcam.com/ps/pdf/protocols/Histone%20extraction%20protocol.pdf

Hi Julie,

I know histones are very tricky to do western on them because of high positive charge and small MW. There are some protocols particularly designed for histone electrophoresis and transfer - buffers contain Triton X100, high urea concentration and acetic acid and specific conditions. Try to look for them in Pubmed.

Good luck

My hesitation with the acid extraction is the dialysis step, which I haven't done since my undergraduate biochem lab. Can anyone suggest a really basic protocol for dialysis?

Hey Julie,

the protocol of abcam I suggested to use is also an acid extraction but there is no dialysis step needed - it is very simple and robust.

Simple and robust protocols are my favorite kind. Thanks, Nora!

you can also use the mini dialysis tubes if u r worried about dialysis..those r so simple to use and no setup required! There is also another protocol without any dialysis...msg me if still interested.

Histone will keep positive charge under regular Tris-Glycine-Methanol transfer buffer. It will transfer to the cathode side instead of anode. You can put another membrane on cathode side. Just use it for Histone western blot.

I have been experimenting the same as you and still does not seem to be good enough.. Julie, did you succeed? I have all the time variable results using THP-1 25x104 cells in one mL re-suspending the pellet in 50uL of lysis buffer and loading 10ug of protein per well. Sometimes it work, sometimes not...The most frequent problem is that I do not have bands in the desired region. At the moment we just have precast gels at 12%. We are a medicinal chemistry lab and I am not very familiar with biological techniques. ANy suggestion will be much appreciated.

Hei Julie, you must be n expert by now....and we have the same problem, any suggestion?

I am using CAPS buffer for Histone protein transfer. This buffer is good for regular western blot and always give me good results for Histone H3 blot (cell signal antibody). check this link for formula:

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6211.pdf

what is the transfer condition for handmade 1.5mm 15% gel to transfer histone H4?

I AM USING 20% METHANOL IN TRIS GLYCINE , 0.2 MICRON NITROCELLULOSE MEMBRANE FOR 100 V HR . the ponceau staining looks weaker compared to precast gel 10-12% with the transfer using sds and ethanol.

please suggest me the exact time to transfer histone H4