I have been running into some difficulties with western blotting. I never see any protein bands in my western blots. I run my western blots in wet conditions and with PVDF membrane. I activate the membrane with methanol before running the transfer. The kaleidoscope (ladder) has been succesfully transferred to the membrane but Ponceau S staining never reveals any bands.
The Laemnli buffer never transfers to the membrane, is this normal? Is the Laemnli buffer supposed to not transfer?
The parameters that I use for transfer are:
-1 hour
-0.4 amps
In cold.
I did the western blotting anyways and no bands were seen, only this in the picture.
The quick and dirty answer is you have a proteinase which ate up your protein reaction. Without seeing the SDS PAGE gel as well it is hard to know. Also your background on that gel seems to be quite high, so if you did get some transfer it would be lost in the noise. Finally, did you remember to use the antibodies? Both the primary and secondary?
Hi
To me 1 hour is quite low ..may be u cud go upto 2-3 hrs since your membrane is not even showing Ponceau stained bands which means there was no trnsfr so you shud try change your transfer conditions. Forget about the use of primary and secondary. if there was no protein on the membrane to begin with what would your pri and sec antibody bind to? Also check your transfer buffer composition especially the methanol content in it.
- The transfer buffer used for western blot is Towbins buffer (pH 8.2-8.4) containing 25 mM Tris base, 192 mM Glycine and 20%v/v Methanol . (It is advisable not to reuse after one transfer)
- Membrane (blot) washing buffer (TBST): 100 mM Tris pH 7.5/ 0.1%v/v Tween 20
- Electrode buffer 5X pH 8.3 (For SDS PAGE): 25 mM Tris base, 250 mM Glycine, 0.1%w/v SDS (dilute to 1x before use)
I used to do western blot using Biorad Trans-Blot semi-dry transfer. The gel and nitrocellulose membrane were intially equilibrated in Towbin’s buffer for 5-10 minutes, before preparing the stack. Electrotransfer was then done at 300 mA constant current for 3 hours, the stack was wetted in-between the transfer with towbins buffer, so it does not dry out. If stack gets dried up, the transfer might stop.
Just make sure your stack is prepared right with correct position of membrane and gel.. and not the reverse way.
After complete hours of transfer, check the gel by staining with CBB-250 and destaining it, no bands should appear if it's complete transfer and also stain PVDF membrane by staining with Ponceau S.. you should see pinkish-orange band on the membrane.
I think you should increase your transfer time.
Also increase your incubation time in blocking buffer (5% w/v in Tris buffer saline Tween 20 (TBST) to get rid of unwanted bands if it appears. Prepare blocking solution fresh (just before use) and make sure the solution is properly mixed and is a uniform mixture with no clumps. Confirm that your blocking reagent is of proper quality (not expiry one).
First fix your primary antibody dilution. If you are getting unwanted bands by adding primary antibody, then decrease dilution of primary antibody depending on the intensity of the band of interest. When intensity of your band of interest is appropriate, it is the proper primary antibody dilution. (fix this dilution when intensity of the band of interest is appropriate).
Then try to standardize dilution of your secondary antibody. If you are still getting unwanted bands then increase the dilution of your secondary antibody until you don't get extra unwanted bands.
Try to maintain temperature of 4°C during incubation with blocking buffer, primary and secondary antibody.
Also confirm that your detection reagents (Reagent A and Reagent B of kits) are not expiry one and mix the two solutions only prior to use.
Good Luck!
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