Dear all,
I am trying to perform a western blot on a low MW protein (~11-12 kDa). However I loose my low MW proteins at the end of the western blot procedure (as shown in picture) and I have no idea why. I tried to dry the blot to "fix" the proteins on the blot without success (same picture). Could anyone help me out? See details of my approach below.
Thank you in advance,
Sjors
- I transfer my proteins from a 4-12% Bolt gel to a 0.2 µm PVDF blot (Invitrogen, LC2002, which I activated in 100% methanol) in a tranfer buffer containing 20% methanol.
- Then I dried the membrane and reactivated it again in 100% methanol (also tried it without drying).
- After a brief rinse with demi H2O I coloured the blot in ponceau S and destained in water (see left picture)
- I completely destained in 0.1 M NaOH (max 30 secs)
- Then I block 1h RT in 5% skim milk (in TBS-T)
- o/n 4°C primary antibody with HRP conjugate (in blocking buffer)
- Wash 4-5 times for 5 minutes in TBS-T
- Incubate with ECL (ThermoFisher 37071) and image
- Briefly wash and stained again with Ponceau S (see right picture)
Use a cellophane sheet below the nitrocellulose membrane during electrobloting which shall allow low molecular proteins to stay on the nitrocellulose membrane
what voltage and how long have been kept the gel for transfer? Low mol wt proteins need shorter time and wet transfer gives better results. May I know the composition of your transfer buffer?
Dear Shamsher and Dharam,
Thank you for the suggestions.
Dharam Singh , I transferred 120 mA (~30 V) for 1h. Transfer buffer concentrations: 250 mM Tris, 190 mM Glycine, 20% methanol, pH 8.3.
I am thinking of fixing the proteins to the blot with paraformaldehyde or Glutaraldehyde, as I read in multiple studies that this was necessary to keep low MW proteins on the blot.
During electroblotting there is a conflict: large proteins need mobilisation (with SDS) to get out of the gel, small proteins need precipitation (with methanol) to stay on the membrane. With semidry blotters it is possible to use mobilising buffers for the gel and precipitating buffer for the membrane (doi:10.1006/abio.1993.1313 ).
However, blotting doesn't seem to be the real problem here, but protein loss during the immune detection. You could try to perform this on dry PVDF membranes (doi:10.1006/abio.1999.4453).
Sjors van der Horst , My experience with low molecular weight protein# is to transfer at 10-15V for 20 to 30min is equally effective without loosing low molecular weight proteins from the membrane in a semi-dry method. Buffer composition seems to be fine...
I transfer low molecular weight protein (~11kDa) in the PVDF membrane using semi dry apparatus at 10 V for 35-40 minutes. Then, I block in a blocking buffer (5% skim milk powder) for 1 hr at RT, followed by O/N incubation with primary antibody. Next day, I wash with TBST for 5 minutes, 3 times.
Is it absolutely necessary to stain the membrane with Ponceau solution? I have used Ponceau to visualize the proteins after the transfer in the past and destained with just the 5% skimmed milk powder in TBST.
Engelbert Buxbaum Thank you for the good suggestion! I will first test paraformaldehyde and gluteraldehyde fixation, but if this does not work then I will definitely try your suggestion.
Gaurav Chandra Gyanwali Thank you for sharing your protocol. It is not necessary to stain the membrane with Ponceau , and I have tried to not stain the membrane after transfer but still the proteins were lost after blocking, primary ab incubation and imaging.
I would recomend you gluteraldehyde fixation. It gave me very good results for very low MW proteins.
Mario Ferrer-Navarro Thanks for the advise! What percentage glutaraldehyde do you use and for how long did you incubate?
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