I load approx 10 ug protein usually for western blotting. the problem that i am facing is that the fresh lysates that are prepared in lysis buffer (Urea, tris base, CHAPS) does not give me a band of interest, rather there are many non specific bands that are observed. . But my old lysate (approx prepared 6 months before in the same lysis buffer) gives me a thick band of interest.The specific band can be presumed to be the one aligning with the old lysate that gives a specific band otherwise in the ladder that is seen my band of interest is of the similar intensity as the non specific ones. This thing happens everytime i put up a western blot.
I have tried increasing and decreasing protein load, changing the secondary antibody, washing conditions, but the fresh lysates do not give me a band. I recently tried with another similar GTPase and it gives me a very prominant band without non specificity. Both the proteins are cytosolic.
The difficulty is that it is not possible for me to homogenize the samples as I have to transfect them in a 12 well plate and extract cells from maximum of 2 wells.. Hence i prefer adding lysis buffer to the cell pellet, overnight incubation at 4 degrees and homogenize using an insulin syringe, pelleting down at 10000 rpm for 45 mins and transferring the supernatant.
Another thing that I feel that my primary antibody works properly is that it detects the band in the old lysate but not in the fresh lysates or mock transfected cells. The primary antibody works fine for immunofluorescence albeit not at a very high dilution.
The protocol that i follow is
10% gel run at 100 V, Transfer at 100 V for 1 hr 15 mins
Blocking with 2% blotto (ECL Advance kit, GE Amersham)
Primary antibody- 1:500 dilution, 4 degrees overnight (Santacruz Biotech)
washing for 1.5 hrs with PBST containing 0.05% Tween 20
Secondary Antibody- 1:12000, 1 hr at room temperature (Swine anti Rabbit-HRP, Dako)
washing for 2.5 hrs with PBST containing 0.05% Tween 20
15 min washing with PBS
Detection with ECL Advance (Ge Amersham)
Help will be highly appreciated.
Dear Ruchi,
is seems to be that you have some proteases in your cell lysate. They are always active, even if it's frozen. They are working much more slowly, but they are working.
Two ways to solve the problem:
Prepare your lysate in the presence of a protease inhibitor (cocktail), PMSF 1 µmol/L could be sufficient for a first experiment and run this against lysates which was stored for 24 h, 48 h and may be longer at 37 °C to achieve a maximum of enzyme activity.
Only if you are using more than 4 mol/L Urea all proteases should be denatured as well. So you can put the cells directly into the sample buffer for the SDS electrophoresis. SDS will lyse the cells anyhow
Check the databases for potential cross reactions of you antibody by the matching the sequences.
Just one short tip: for the suppression of mild cross reactions, you can increase the salt concentration in the incubation buffer (phosphate buffer, ph 7.2, 0.3 mol/L NaCl, 0.05 - 0.1% Tween 20, 1 - 3% protein like BSA, HSA, ...)
Hi, you may try to have a positive control or purified protiein of inerst and check for the precise band.
It should not be a problem if you see other non specific bands, if you are sure that your protein of interest may not have been degraded or fragmented. Further, if you are not concentrating the sample for endosomes, try to add Laemelli buffer directly into the well, scratch the surface, collect, boil, load to gels and see if your band is clear.
Dear Sir, as i mentioned, usually the old lysate serves as a positive control everytime because the band of interest is of similar intensity as rest other non specific bands and hence difficult to recognize in absence of a positive control.
Another thing, that the protein of interest expresses well in the OLD lysate.. Being an enzyme I really dont know how it survives repeated cycles of freezing and thawing. The fresh samples fail to give me the specific band.
I did not understand your last suggestion. if you could be kind enough to explain it to me in detail and the reason for doing the same..
Thank you..
Dear Gael, The protein is constitutively expressed, nothing of the transient sort.. In fact it used to give a really thick band when I had initially started Western Blotting with the same some 2 years ago.
Dear Kathrin,
1. It is a constitutive protein.
2. Primary antibody is polyclonal from rabbit (dilution 1:500), secondary antibody is swine anti rabbit (dilution 1:12,000) and the detection system is ECL advance from GE Amersham.
3. I do not reuse the antibody. Freshly diluted antibody is used for every experiment.
Dear Ruchi,
is seems to be that you have some proteases in your cell lysate. They are always active, even if it's frozen. They are working much more slowly, but they are working.
Two ways to solve the problem:
Prepare your lysate in the presence of a protease inhibitor (cocktail), PMSF 1 µmol/L could be sufficient for a first experiment and run this against lysates which was stored for 24 h, 48 h and may be longer at 37 °C to achieve a maximum of enzyme activity.
Only if you are using more than 4 mol/L Urea all proteases should be denatured as well. So you can put the cells directly into the sample buffer for the SDS electrophoresis. SDS will lyse the cells anyhow
Check the databases for potential cross reactions of you antibody by the matching the sequences.
Just one short tip: for the suppression of mild cross reactions, you can increase the salt concentration in the incubation buffer (phosphate buffer, ph 7.2, 0.3 mol/L NaCl, 0.05 - 0.1% Tween 20, 1 - 3% protein like BSA, HSA, ...)
Sorry, I have no idea why you are having urea in ur lysis buffer. Is ur lysis buffer freshly prepared?.
Like many other people suggested, the best idea to exclude problems in lysis or degradation during the processing is to lyse directly in SDS loading dye (with DTT/mercaptoethanol). We use it generally in 2x concentration and adding this directly to cells will lyse and release most of the proteins (including keratins etc etc). You can mix them by pipette, collect to a tube and boil directly for 5-10min. Then just give a short spin and load from top.
Hi, I meant about directly adding Lamelli buffer (also sometimes denoted as loaidng/sample buffer) to the well, in your case 12 well plate, to lyse and extract the sample for WB direclty, without undergoing sperate lysis step, then adding a loading buffer, before running onto gel. By this you also avoid, any chance of protease reactions. I think this is also similar to the suggestion by Stephan & Manoj, to directly put cells in to sample buffer. If you dont have any choice, but lyse cells first with lysis buffer, before adding loading/sample buffer, then try including protease and/or phosphatese inhbitors to the lysis buffer (as suggested by Stephan).
But I still do not understand, why you got the band before, and not now. I guess you would not have added them (protease inhibitors) before too, in case if you are not adding it now. Thats why, I suggest you to still go for a positive control, also to confirm that the band which you saw in the old sample is really the band you needed. I think, you could rely on the band at appropriate position, as per the molecualr weight, until there was no other band, or controversy related to that.
One more suggestion, though I doubt it may solve the problem is to try to use a monoclonal ab, if available.
I agree with Stephan and Vinoth. It seems to be a problem of protein degradation. You should add PMSF, EDTA and protease inhibitor cocktail to your buffer (there are several commercial cocktails with different compositions). Please ensure that the solutions are properly stored and not expired.
Dear All,
As per your suggestion about the protease inhibitor, I have previously added PMSF to one set of samples but still could not see the band of interest there..
If you do not have protease inhibitor cocktail, load protein directly into sample buffer. Also it is not wise to prepare fresh antibodies for every reaction run. It is waste of antibodies as well as against the rules of optimization. Don't freeze & thaw stock antibodies again & again. Store diluted antibodies at 4oC and use them for several times.
To all above comments I would also add that using your old extracts as a positive control just because it gives you a strong band of the correct size doesn't make much sense. If previously this extract didn't show this band, then probably it is still not your protein of interest but products of degradation of bigger proteins. To be absolutely sure this is the correct band you can use a blocking peptide (your primary is from Santa Cruz, I think they supply blocking peptides for most of their antibodies).
Also, I agree that using a protease inhibitor cocktail is an absolute must. We also use PMSF, SOV, EDTA plus a cocktail.
As to the many unspecific bands you see, try to increase salt concentrations in your PBS. Try also other blocking solutions: non-fat milk or BSA.
Do you detect using a traditional film or a CCD camera? How long do you have to expose to see signal? If the exposure time is really short, try decreasing primary antibody concentration. Too high will also produce many unspecific bands.
Hope that helps.
Kris
IF I understand corectly, u get a lot of unspecific bands. I would sacrifice a day to test the secondary antibody -use it on the membrane without the primary ab.
Helo dear,
Try to check the sequence of your clone that stop codon may have appeared. you can check the sequence by sequencing it.
Dear Ruchi, u need to prepare whole cell lysate by good lysis buffer(tris, edta, egta and triton-x-100) and use ice cold solubilisation agent (check in paper)which can solubilise yr protein of intereet better. load around 15-20 ug of protein in sds.
Dear Kris,
The lysate that serves as a positive control was prepared about 6 months ago and stored at -20 degrees and not thawed and frozen till the time it was used for blotting experiments so i presume the positive signal is not the degradation product of higher molecular weight proteins. and everytime that the lysate was used it gave a positive signal.
now i will try increasing the salt concentration in PBS as you suggest.
the detection system that i use is ECL advance from GE amersham. it usually takes about 30 sec to a minute to give me the chemiluminescent signal. the positive control lights up at the specific position whereas with the other samples the background starts fluorescing. so fiinally at the end, the positive control gives a specific band with very few non specific bands due to high exposure time and other samples end up giving me a ladder like pattern where i am unable to make out my specific band of interest..
Dear Ana,
As you say, i load a negative control too everytime without the primary antibody using one of the samples. it usually gives very few (one or two) higher molecular weight non specific bands.
Dear Arpita,
I am just trying to optimi tze western blotting experiments with just my lysate. transfection is something that i would do but later on. so just need to work with my western blotting experiments at first
Dear Jyotirmaya,
I ll go through certain papers to check out lysis buffer composition. what would you suggest for RIPA buffer. is it worth trying?
Dear all,
Thank you so much for your valuable inputs. I am really happy that all of you are sharing your suggestion and also showing a keen interest in my problems. Heartiest thanks to all of you
RIPA buffer also good. make sure that your protein of interest is well solubilised or not. then it will give good resolution bands.
Dear Jyotirmaya.
The protocol that i usually follow with lysis is suspending the cell pellet in lysis buffer, vortexing for a minute, overnight incubation at 4 degrees and the spinning at 10,000 rpm for an hour, transferring the supernantant to a fresh tube and using that supernatant.. i will follow the same protocol with RIPA buffer. if you have any additional suggestions to it, pl let me know.. thank you.
The way we do it with RIPA is to suspend cell pellet in RIPA, vortex, incubate on ice for 30min, vortex again, and then centrifuge at 10,000g to pellet debris. Then you can proceed to measure protein concentration. Remember not to use Bradford with RIPA, it's incompatible.
- Badges
- Science method