I am following the same steps for transfer method but my proteins aren't getting transfered to the membrane only the marker is getting transfered not my proteins. Could you please help me in this as I'm trying it from a month.
Actually the size of my protein is 43KDa and 47KDa. High molecular weight proteins are easily transferred but issue is with low molecular weight proteins.
We have this turbo transfer system but I do not have the pre-cut transfer pack. What is the soluble used in the pre-cut transfer pack to soak the membrane and filter papers? Is it just transfer buffer without methanol? I'm trying to save money by cutting my own membrane and filter papers. Thanks
Towbin Buffer 25 mM Tris, 192 mM glycine, 10% ethanol is fine (do not adjust pH). CAPS Buffer CAPS-based transfer buffer (10 mM CAPS, pH 11, 10% ethanol) may be preferable for transfers of high molecular weight proteins (for example, >150 kD) and in cases where the glycine component of Towbin buffer may interfere with downstream protein sequencing applications. Dunn Carbonate Buffer 10 mM NaHCO3 (sodium bicarbonate), 3mM Na2CO3 (sodium carbonate), 10% ethanol is fine Ethanol necessary ONLY FOR PROTEIN LARGER THAN 20Kd SDS only for protein larger than 80 KD up to 0.1% (start at 0.03%). For large protein use a 6-7.5% (easy to break) gel or gradient gel. PREWETTING PVDF MEMBRANE WITH 200 proof ethanol is as good as methanol.
Let me know, at this point I've given up, and gone back to the old school tanks... takes forever but works, would love to get the turbo going... let me know what you find out.
@@SunSurfSnow1990 Dip the roller in the buffer solution and apply it to the membranes, sometimes when they get dry that happens, at least that's what we did and it worked
I am following the same steps for transfer method but my proteins aren't getting transfered to the membrane only the marker is getting transfered not my proteins. Could you please help me in this as I'm trying it from a month.
did you find out why? I also get partial transfer
Actually the size of my protein is 43KDa and 47KDa. High molecular weight proteins are easily transferred but issue is with low molecular weight proteins.
add more methanol into your transfer buffer@@aynalsaba7982
I want to know after incubating with 2nd Ab, how long should I wash? And how many times?
it depends. Typically you wash three times with TBST 10-15 minutes each.
We have this turbo transfer system but I do not have the pre-cut transfer pack. What is the soluble used in the pre-cut transfer pack to soak the membrane and filter papers? Is it just transfer buffer without methanol? I'm trying to save money by cutting my own membrane and filter papers. Thanks
Transfer buffer with 10% ethanol.
Towbin Buffer
25 mM Tris, 192 mM glycine, 10% ethanol is fine (do not adjust pH).
CAPS Buffer
CAPS-based transfer buffer (10 mM CAPS, pH 11, 10% ethanol) may be preferable for transfers of high molecular weight proteins (for example, >150 kD) and in cases where the glycine component of Towbin buffer may interfere with downstream protein sequencing applications.
Dunn Carbonate Buffer
10 mM NaHCO3 (sodium bicarbonate), 3mM Na2CO3 (sodium carbonate), 10% ethanol is fine
Ethanol necessary ONLY FOR PROTEIN LARGER THAN 20Kd
SDS only for protein larger than 80 KD up to 0.1% (start at 0.03%).
For large protein use a 6-7.5% (easy to break) gel or gradient gel.
PREWETTING PVDF MEMBRANE WITH 200 proof ethanol is as good as methanol.
I have this unit, but the run wont go above 0.1A... why could this be?
Let me know, at this point I've given up, and gone back to the old school tanks... takes forever but works, would love to get the turbo going... let me know what you find out.
@@SunSurfSnow1990 Dip the roller in the buffer solution and apply it to the membranes, sometimes when they get dry that happens, at least that's what we did and it worked
Sometime it depends on the constant A or constant V run.