Hi,
I am new to western blotting and so facing the problem of blank blots.
i want to assess the expression of two membrane bound proteins, 45 & 74 KD, in rat heart tissue. My tissue samples are stored in liquid nitrogen.
My protocol as follows:
Grinding 60-80 mg of tissue in liquid nitrogen using mortar and pestle
incubation of tissue powder in RIPA buffer + protease and phosphatase inhibitor cocktail for 1 hour at 4 C, with needle shearing using 21 guage needle.
centrifuge at 22,000 xg for 20 min, collect the supernatent and discard the pellet.
Denaturation of proteins was done using 2x lamaelli buffer and boiling at 95 C for 5 min.
loading 40 ug / well of the total protein and using 10% SDS-PAGE.
transfer of protein to nitrocellulose membrane, and assaessing effiency of transfer using ponceau s stain
blocking membrane in 5% non fat dry milk, overnight
incubation with primary antibody, many different dilutions, overnight at 4 C
incubation with secondary antibody for 2 hours at room temp.
colorimetric detection using TMB substrate solution.
after this, I always gt a blank signal with my two membrane proteins.
noteworthy, I had a signal when I probed the membrane with actin antibody. but this signal washed away in the substrate solution with continuous shaking, leaving also the membrane almost blank, and turning the color of the TMB substrate sol. into light blue
My primary antibodies does fine in IHC, so I do not think that they are blamed
My secondary antibody gives a deep blue color when I incubated it with TMB sol. in an eppendorf.
I am not sure whether the problem is in my protein extraction protocol or in one of my antibodies, including my secondry, or in the TMB substrate sol.?
It may be related to the transfer step..... What is your transfer method?
There are a number of possibilities here. First, if even your positive control washes off in the substrate solution, then it's very likely that your targets are also washing off in that solution. Colorimetric detection isn't very sensitive; can you use fluorescence or chemiluminescence? First fix the problem with your substrate solution washing off the target; if you can't see even your positive control, you can't be sure that any other suggestions will work (or that you'd know if they did).
Membrane-bound proteins can also be problematic in Western blotting, much more so than intracellular proteins. Additionally, actin is highly expressed; are your targets highly expressed in this system? You could use one of the many protocols that collect samples that are enriched for membrane-bound proteins and adjust your protein concentration to increase the amount of your target protein.
Have you tried different blocking buffers? Milk is a good one, but there are others that the antibody may work better with. Is your blocking buffer percentage too high? Some antibodies show up better after 1% milk block than after 5% milk block.
Have other people used this protocol successfully to look for your target proteins? Which protocols did work successfully for other people? Which protocols are available to you, and do those include the protocols that worked for other people in the past?
What is your transfer method? Have you tried transferring to PVDF membranes instead of nitrocellulose? What voltage are you transferring at?
Hi Nehal Mohammed Ramadan
Are you using TMB in WB? The final product in TMB reaction is soluble, so you cannot use it in this kind of assay. TMB is intended for ELISA not for WB. You may use DAB, 4Cl1N or AEC. All of them form an insoluble product that is appropriate for WB.
good luck!!!
Just as J. Corthell said, W.B. assay has many steps that might go wrong. Firstly, as far as the lysates are concerned:every time I have a problem that points out to the lysates preparation procedure I use a simple 2% SDS 60mM Tris HCl ph=6,2 buffer instead of a RIPA+Inhibitors cocktail. Note that you have to boil the lysates for 10min with this procedure and then they are ready to be used. Although Western blott-ing membrane proteins is a little bit tricky, so I suggest you search for a tailored WB protocol. Secondly, are you sure your gel is ok and runs ok? Does the Ponceau show dense bands in all your lines? And are you sure you are washing the dye enough before beginning blocking the membrane? I use Ponceau after concluding the whole procedure, and only if my blot is "tabula rasa". This way I am sure both running and transfer are correct and efficient. Another way to ensure this is by dying your gel with Coomassie after transfer in order to see what is left behind. Thirdly, are you doing a semi-dry or liquid transfer? In general I suggest the latter.Are Amperes stable and not Volts? In how many A are you transferring?(you should also take into considerations the volume of the transfer machine to compute the A). Moreover, is your blocking buffer fresh? Old blocking buffer tends to present precipitates, usually mucus. If your blocking is contaminated then there is the possibility that mucus/bacteria substances interact with the membrane blocking Ab sites. To prevent this I use 0,05% sodium azide(toxic) when diluting my Abs, to block metabolism. Moreover, the best blocking buffer is the one that works best for your Ab. So check what the Ab data sheet suggests and act accordingly. Also, what is the ideal Ab concentration for WB? ICH and WB usually demand different dilutions. Check the Ab data sheet once again. Are you sure your Abs are for WB on the first place? Because there are many that do not. If your Abs have precipitates like th ones mentioned above (since they are not fresh dilutes) spin down the precipitate and relocate the diluted Ab in a new falcon in case you do not have much Ab stock. Is your secondary Ab HRP-conjugated?Or is it compatible with the substrate -enzyme system you are using? TMB as Oscar Otero said is usually for Elisa. There is however a Sigma Chem specific for WB. There is also another question in research gate about it (https://www.researchgate.net/post/Can_I_use_ELISA_TMB_substrate_for_Western_blot) Are you sure you are using the correct stuff? Furthermore, are you sure the amount you use covers the membrane while shaking?
Hi, thank you for all your valuable answers.
I am using a wet transfer method, with 350 mA for 90 min, in an equipment that takes around 1.5 liters of transfer buffer.
My primary antibodies are applicable in WB, and my secondary is HRP linked. Dilution of ab(s) are chosen according to data sheet, also the blocking sol. The difference is that they report blocking for 30 min and I did it for 24 h, could this affects the result?
Many references report using of RIPA buffer for extraction of my markers from mem. of cultured ventricular cells, and on reference report using the same buffer with ventricular tissue samples. So, I should ask whether this matters?
some references report using a protocol that involves ultracentrifugation for separation of mem. fraction. unfortunately, we do not have such ultracentrifuge in our college.
My proteins of interest belongs to connexin family and subunit included in the structure of potassium channels.
my protein yield is about 15 mg/ml extracted as mentioned from 60-70 mg of tissue using 400 ul of buffer. Here, I should ask if using the tissue tearor would increase the protein yield from membrans?. Also, if longer incubation with RIPA could increase the mem. protein solubilization?
And of course, I am using a recipe of soluble TMB sol. with the addition of EDTA. I do not know if the EDTA can act as a precipitating agent, but it works well with my supervisor, as she says.
However, I have a 50X DAB concentrate, can any one help with a recipe to use it for colorimetric detection, as this is the only detection method available.
Attached is my mem. stained with ponceau, as I do not know what the meaning of " tabula rasa" dear Nktaria
ahahaha I am surprise that somebody use "tabula rasa", it is Latin. In Italy we use this expression with a different meaning.
All suggestions are right, I would like to ask to Nehal, is your protein from a housekeeping gene? As you have detected the actin, you can assume how much of your protein you should have in your lysate and decide whether you need to load more proteins in your gel or to change antibody.
As Nektaria M. Leli mentioned, your primary antibody may not be suitable for western blot. Our lab has produced numerous monoclonals that bind well in ELISA and RIA, but have never shown a signal when used in a Western. We have actually had better luck using antibodies purified from ascites fluid, which gives a polyclonal product. I would try another antibody if available. Also, we're very happy with GE Healthcare's ECL Plus as a developing agent for chemiluminescence, if you have that option available.
Hello Nehal:
A lot of good suggestions have thrown by different people. I have couple more.
1. Adding protease and phosphatase inhibitors during the protein extraction step.
2. Increase the total amount of protein from 40.0 to 60.0 or even 100.00 ug/lane.
3. Activate the PVDF membrane in 100% methanol for 1-2 min and then in the running buffer for 5-10 min before placing them in the sandwich. After placing all the other stuff and before closing the cassette, roll over a 15 ml tube gently across the sandwich and then close it. I helps a lot.
4. Try to run the transfer phase at a slower voltage/miliamp but for a longer time (if you are a college going student that becomes a problem, not much time to do research).
5. Try to avoid milk as your blocking buffer, instead you can use Tween-20. Milk can block all phospho-proteins specially. So all together I stopped using them..
6. Concentration of primary as well as secondary antibodies matter. Check your datasheet and if needed talk to the tech services of the company and that's why they are for.
7. Alk-p conjugated secondary antibodies work good for me and I use a NBT-BCIP color detection system.
Hope it helps..Any further question? Write again..Good luck.
Dar Paola,
I do not think that my proteins of interest are coded with housekeeping genes. Connexins are widely expressed in different tissues, but in many different isoforms. Voltage gated potassium ion channels are expressed mainly in excitable tissues, including heart tissue.
Since you have used these antibodies with IHC and they were fine, I would recommend to spin at 48,000 xg for 20 min and take the pellet instead of the supernatant. Using this speed you are going to isolate the sarcolemma vesicles in which you will have your protein integrated> hope this will help.
Regards
Dear Nehal,
Othman has a point, look in the pellet for your protein. What you also could try to get it soluble is the use of CHAPS at around 1% final concentration, you could even just add it to your RIPA. It is a zwitterionic detergent aimed at extracting membrane proteins, worked well for me when isolating protein from tumor tissues.
Best,
Kevin
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