- Maathangi Murali added an answer:18Does anyone have experience in TNF-alpha detection: ELISA vs Western Blots?
For my MSc I wanted to determine the levels of TNF-a released by H9c2 cells in response to treatment with Doxorubicin. I treated every day for 5 days, so after each day I collected the cell culture medium and froze it quickly. On day 6 I did the ELISA. Most of the values O obtained were almost identical to the control, but I did get significant increases at a few groups (see graph). I then wanted to confirm this with western blotting, but now I see an increase in TNF-a as soon as DOX is given and it increases as the concentration of DOX increases. I would like to know why my ELISA and Western blots are not producing the same results and how I explain this? Thank you
ELISA is the best optionFollowing
- Natascha Fussi added an answer:9Does anyone have a good protocol, or tips to perform a Western Blot with cell supernatant samples?
I tried several times to perform an alpha synuclein Western Blot with cell supernatant samples, but I could not detect the normal (16 kDa) band. I used to concentrate the medium before with Centrifugal Filter Units from Milipore. Does anyone have any suggestions?
Thank you for all of your answers.Following
- Ketil Winther Pedersen added an answer:53Having issues getting primary antibody to bind during Western blots involving exosomes.I have been successfully performing Western blots on cell lysates from human glioma tissue using tags for actin, HSP70, HSP90, and others, however, I cannot get the signal to show up when I include lanes with exosomes. Even when I alternate lanes (cell lysate, exosome lysate, etc.) I get nice crisp bands in the cell lanes but not in the exosome ones.
I have tried Laemmli 2x buffer as well as RIPA buffer. Transferring the proteins from gel to PVDF always works, for both cells and exosomes, and I get clear transfer in exosome lanes at the MW that corresponds to HSP70, which is what I'm most interested in.
i will send you some of our work and protocols by mail.
- Maathangi Murali added an answer:4What happens if I store mammalian cells with loading buffer in -20 (western blot samples)?
What happens if I store mammalian cells with loading buffer (western blot samples) in -20? I forgot to boil the cells before freezing. Could I boil them now and continue my experiment or I should repeat the experiment?
Depends on cell type and how long it is stored also if you would have added some chelating agents then it would be fineFollowing
- Leslie kay Morgan added an answer:7Any tips to western blot a 10 kDa protein?
I am trying western blot for an over-expressed FLAG-tagged peptide of 10 kDa with a pI of 7.8. I can see it is expressed on a dot blot, but I have yet to be able to successfully transfer it.
I run 4-20% tris-glycine gels from Bio-Rad with standard laemmli sample buffer. I use a bio-rad power supply, wet tank transfer apparatus, and towbin buffer pH 8.5 with 20% methanol. I have tried both a 90V transfer for 1hr and a 30V overnight transfer at 4C. I thought I might be over-transferring and cut back the time on the 90V transfer to 15 and 30 minutes with no change.
My next thoughts are to drop the overnight transfer to 20V, remove the methanol as some say it can hinder transfer, up the methanol as Millipore suggests 35-40% for transferring LMW proteins onto PVDF, or switch to 0.2um nitrocellulose as bitesizebio.com says it is better for <20 kDa.
If anyone has experience to inform the direction I should head, I would much appreciate your advice.
These are all good suggestions. To make sure your protein is still on the gel run an extra lane that you do not transfer. Instead cut off the extra lane and stain to make sure your protein has not run off.Following
- Nino Kvirkvelia added an answer:4Can we do fluorescence western blot to the same membrane we used with ECL?
In my lab we are currently starting to use li-cor fluorescent western blot and one of the informations I coudn't quite grasp is that we shouldn't use the membranes already used with traditional ECL. So true or false? Is this really a thing and we might need to stripp the membranes before switching to the fluorescent approach or is some misguided info?
Thanks in advance
If you want to avoid stripping of secondary and primary abs you could try to use fluorescent labelled "anti-scondary" abs, after removing ECL.Following
- Karolina Nowicka-Bauer added an answer:1Does anyone know that Mem-PER Eukaryotic membrane protein extraction reagent kit (Thermo) can be used for bacterial membrane proteins extraction?
I am trying to extract membrane bound proteins from E.coli for western blotting by using AP detection method.
However, I cannot find any commercial reagent kit regarding bacterial membrane proteins extraction.
Therefore I wonder that whether Mem-PER Eukaryotic solution kit can be applied for this ?
Thank you so much in advance for your kind help.
I know this kit very well :) I think you can use it but my experience is that I have never obtained real phase separation. I have tried many many times. I had no problem with this using traditional protocol for phase separation (with TX-114). I have tried kit from Bio-Rad where you have to use sonicator. It worked well and think you would have no problem with bacteria :) btw, I know this kit from Thermo is no longer for sale (at least 3 years ago) so probably yours is out of date but if it worked earlier it still should work.Following
- Chithan C Kandaswami added an answer:6What does lectin bind to on a blood vessel?
Lectin (Lycopersicon esculentum lectin) is the most widely used tool to visualize blood vessels. It is known that lectins bind to glycoproteins located in the glycocalyx and in the basal membrane of endothelial cells. Does anyone know its exact specific molecular target? Has anyone performed Western blot using lectin as a probe? If yes, at what molecular weight/s can we expect lectin reactive (lectin-specific) bands (for example in brain lysates)?
I could find one reference which states that, "... lectin is specific for
(GlcNAc)n (n >3) and poly-N-acetyllactosamine residues (Nachbar et al., 1980;Zeng et al., 1998)"
However, this does not clarify the identity of the target molecule to which lectin binds. Please find the attached article citing this reference
<<It is known that lectins bind to glycoproteins located in the glycocalyx and in the basal membrane of endothelial cells. Does anyone know its exact specific molecular target? >> Lectins from tomato bind to oligomers of glucosamine.
Lectin activity was described some 150 years ago, when cell agglutination by snake venom and hemagglutination (the clumping of erythrocytes) by castor beans were described. Lectins are proteins of nonimmune origin containing one or more catalytic domains, which reversibly bind to mono- or oligosaccharides. Some lectins binding monomeric mannose are also known. Certain mammalian serum glycoprotein glycans possess terminal sialic acid residues; upon cleavage, these residues expose galactose residues, which bind to asialoglycoprotein receptors, which are C-type galactose specific lectins. Selectins, C-type lectins (see answers above) facilitate the adhesion of leukocytes to endothelial cells of blood vessels. This adhesion is caused by selectin-carbohydrate interaction. Lectins with low affinity to bind to a monosaccharide can bind with much higher affinity to an oligosaccharide containing two or more monosaccharide residues in a favorable linkage. Lectins can bind simultaneously to more than one oligosaccharide. The biological origin and structure of lectins influence the nature of sugars to which they bind.
Selectins, constitute is one of the families of cell adhesion molecules present in the endothelial glycocalyx, a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Glycoproteins from the selectin family possess a terminal lectin domain, which is mainly responsible for the binding of carbohydrate groups on glycosylated proteins or lipids. The glycocalyx of the aortic endothelium recognizes the Wisteria floribunda lectin, which binds N-acetyl-D-galactosamine, while the lectin from Lycopersicon esculentum (tomato) exhibits an affinity for N-acetyl-β-D-glucosamine oligomers.
Western blot analysis of isolated endothelial cell membrane glycoproteins. Schumacher U, Vischer P, Völker W, Engelmann B. Lectin binding and uptake and glycoprotein characterization of isolated porcine aortic endothelial and smooth muscle cells. Cell Biochem Funct. 1993 Sep;11(3):225-30. PubMed PMID: 8403237.Following
- Antonietta R. M. Sabbatini added an answer:9Does Ponceau S staining effect the binding of the primary and secondary antibodies in western blot?
im doing a western blot and want to know if ponceau s staining of the nitrocellulose membrane can effet the binding of my primary antibody if the red stain is not washed out completely with TBST.
Cheers and thanks!
In my experience I never had problems doing Ponceau S staining before the Western blot. protocol.Following
- Masood Sepehrimanesh added an answer:4Can anyone recommend a protocol for the extraction of total protein from a parasitic nematode?
I am working with a parasite who total protein contents I have to analyse both by PAGE and Western blot. Can anyone suggest a protocol for going about this. I will be grateful. Thanks
It is better to use PBC 0.01 M, pH 7.4 and use of Mortar and Pestle for homogenization of the tissue under liquid nitrogen. Then centrifuge the hemogenate at high speed and precipitate other materilas by TCA/acetone method.
I use it for several tissue and found reliable results.Following
- Nicolas Luigi Pascal Casadei added an answer:5Any advice on a BCA protein determination assay that is over-estimating protein?
I am working with total cell lysates in RIPA buffer (Nacl, Tris, NaDeox, triton, SDS, PMSF, protease inhibitor cocktail, Na Orthovanadate, glycerol and NaF). To estimate the total protein concentration in the samples, I use the BCA assay from a kit. After loading the supossed same quantity of protein in each well and making a western blot, I see really big differences between the different lanes. One suspicion that I have is that I have a high quantity of phospholipids in my sample, but I'm not really sure. Any advice is really welcome!
I do not think that the BCA measurement is that bad. Honestly, we all made dilution raw to make the estimation and I am used to see regression coefficient above 0.98 even if you take a standard diluted in RIPA buffer (Form Adam).
You might reconsider the homogeneity of your samples (which lysing method do you use? Are you using homogenate or lysate?). I would rather think that you have a problem of loading control (probably not really adapted). Did you tried reversible staining dye such as ponceau to confirm the problem?Following
- Kaarel Kõivupuu added an answer:11Does anyone have experience with a western blot gel polymerization problem?
After running several Western blots I ran into a problem that I didn't seem to find an answer to from here. The gels polymerize evenly except for the bottom 2-3 millimeters - if I remove the plate from the stand a little bit of liquid drops off, leaving a rather ugly zigzag looking bottom edge. It's not a big problem, but rather unfortunate as I need a clear view on smaller peptides on my blots as well and a clear edge on the gel would definetly help.
To avoid some question - I have new APS, roughly a month old acrylamide stock that I keep in the dark (and cold) and the separating buffer is roughly of the same age and should be in order. TEMED might be a question mark, though as much as I understand it works only as a catalyst and should play a role here (in case of a problem with TEMED I should get a bad gel in general). I wash my glassware every time as well. The gel that I'm trying to run is 12%.
Acrylamide-bisacrylamide ratio is 37.5-1.Following
- Eiji Kinoshita added an answer:45Accuracy of Phos-tag gelsIs anyone working with Phos-tag gels? I have just started out working with them and I understand that with such gels, the protein marker is not an accurate indication of the weight of the protein of interest. However, after blotting for my protein interest, it seems that the darkest bands seen are in the range of 25- 35kDa where in theory, it should have been seen at around 60 - 70kDa. Is this some non specific binding of the primary Ab or is there more to the phos tag gel that I do not know about.
I usually use a RIPA buffer, for example, consisting of 50 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 0.25% (w/v) sodium deoxycholate, 1.0% (v/v) Nonidet P-40, and 1.0 mM EDTA. And in the buffer, 1.0 mM phenylmethanesulfonyl fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 µg/mL leupeptin, 1.0 mM Na3VO4, and 1.0 mM NaF are further added before use as a lysis buffer. Commercially available Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail can be also used for Phos-tag SDS-PAGE. It’s no problem at all as far as the use of common-sense concentrations of them. An important thing is that their compositions are the same in all the samples for the analysis of Phos-tag SDS-PAGE.
In the Phos-tag chromatography method, inorganic phosphate or pyrophosphate is used as an elution buffer. I think that the manufacturer will recommend so that to avoid a leak of phosphorylated targets.
- Hyo Min Kim added an answer:3I keep trying Western Blotting using lysozyme antibody (from Santa Cruz and abcam), however I couldn't detect the band. Any suggestions?
I keep trying Western Blotting using lysozyme antibody from SC and abcam, however I couldn't detect the band. Is there any suggestion that I can get through this?
I used donkey anti-goat for a secondary antibody as recommended in the data sheet. I also used a method of HRP staining and tried to detect the band using LAS.
Thanks to Victor and Brandon for great comments!
Sum up the comments, i need a positive control for Lysozyme Ab.
The amount of protein was enough to detect the band, and also using pi. Yes, i have done the ponceau after transfer also..
hmmm let me first try with the positive controlFollowing
- Camille Hallez added an answer:6Do you know a good antibody against HBV capsid for western blotting ?
I am currently searching for a good antibody to detect HBc proteins in western blotting (and why not in immunofluorescence). I tested an antibody from Santa Cruz but I'm not really convinced. For now, I especially need something for western blotting to prove that I do have viruses after purification (I'm interested in proteins inside the capsid, I detect them in WB, but i lack the control...)
Thanks for your help !
Thanks for your answers. I tested the Anti-Hepatitis B Virus Core Antigen antibody [C1] (ab20523) and I have nothing in WB, even if I use Femto ECL or little diutions (I tried 1/50 for example). Actually, I want to detect viral particles produced after transfection of pCayw, and not after infection. I tested the Abcam antibody and 2 Santa Cruz (one against HBc and the second one against pre-S1) and I never detect the virus, whereas I do have viral DNA detected in qPCR (I precipitate the viral particles and then I extract DNA or do a WB). So if one of you has a good solution, I'll take it !
Tian Xia, thanks for your help. I'm gonna test it, I wanted forst when I saw litterature but somebody in the team told me it was not that efficient in WB... Moreover, I can't find informations about its price on the Dako website.Following
- Ishtar Alethari added an answer:7Why do I get two bands for haptoglobin in western blot?
I looking for haptoglobin in mice samples by western blot and I found two bands appear in the membrane and I want to know is that correct or need to check again , and the interestingly same two bands appear for 35 samples.
Thank very much for all you to a useful answers and I will try to change the antibody for same samples and see the results if it same or different.
Best wishes to allFollowing
- Chiara Benedetto added an answer:6Problems with RT-PCR (shRNAs testing)
I am testing some shRNAs by RT-PCR and I do not have hands-on experience with this technique.
In particular, I tested some primers and a PCR reaction for the very first time, but I obtained the expected signal just for the positive control (without shRNA). Before starting any experiment, I made sure that primers sequences were correct, as well as amplicon size, and I set an annealing temperature based on primers Tm.
I cannot justify my result by saying that all my shRNAs are working, because I do not have any signal in my shRNA control! What can I do to optimize my RT-PCR?
Many thanks for your answers.
actually the housekeeping gene yields the same signal in all the samples. What I did not manage to see was the signal in the "empty vector", but now I've optimized my RT-PCR and it works.
Thanks again for all your useful suggestions, they helped me a lot! And good research!Following
- Rongqing Aaron Pan added an answer:99+Western blotting - using BSA or milk? And TBS-T or PBS-T?I want to detect phospho-proteins as well as full-proteins. Does anybody have experience with using BSA instead of milk for blocking and using TBS-T instead of PBS-T?
For WB with phosphoprotein, I think the most important thing is to dilute your primary antibody with BSA solution.
The primary phosph-antibodies might be overwhelmed and sequestered by The phosphor-proteins in milk/Casein.
While it sounds reasonable to use BSA for blocking, sometimes, you might get a high background due to insufficient blocking. In this case, you might think to block with milk which has stronger blocking abilityFollowing
- Simone Tamburri added an answer:8Is SDS an absolutle killer to HPLC-ESI-MS analysis of protein bands?
Hi, several people I know notified me that if I want to cut out protein bands from SDS Page gel and use them for HPLC-ESI-MS analysis, it is absolutely necessary to use precast gels. Is it true? If it is, is it because of the SDS that I would normally use in SDS-PAGE?
The problem with mass spectrometry a part keratins are DETERGENTS. But for sure it is not a problem when you work with gels (because the gel step is done to clean up your sample: It exists also in soluton digestion but then if you have detergents there you will through away your sample). So don't be worry. MS spec could be done with both home made and precast gels (depends from the complexity of your sample and from how much you wish to separate your mixture). Make all solutions clean, put always glowes and labcoat.Actually we have also a facility so we can keep in touch.
- Colin Germer added an answer:5Does anyone use the iBlot gel transfer system?
We are trying to perform Western blots looking for a 1KDa protein and can't seem to find any bands in that range. The smaller bands on our ladder don't seem to transfer well when we image them, would a wet transfer be more effective?
I use P3 for 7.Following
- Kimon Lemonidis added an answer:4Why am I seeing a band that is much higher in kd than the predicted for my protein of interest?
I ran a western to test our GABA alpha 2 and 4 antibodies on (P1) rat brain tissue lysate. GABA alpha 2 was a success with a band at ~51kDA, but I am experiencing a problem with GABA Alpha 4 (Millipore AB5457). The predicted molecular weight for alpha 4 is 64kDa, but after a 10 minute exposure with ECL, there is only one distinct visible band at approximately 110kDa, and nowhere else.
The protocol for my western is:
1. Electrophoresis @ 150V until sample clears well then increase 200V for ~5hours @ room temp
2. Transfer was done overnight on 25V at room temp.
3. Block 1 hour in 5% NFDM
4. Rinse 3x with WB
5. Hybridize with primary antibody (1:500) overnight (~20hours) @ 4C on shaker
6. Rinse 3x with WB
7. Hybridize with HRP-secondary antibody (SC-2004) (1:5000) 1hour @ RT on shaker
8. Rinse 3x with WB
9. Develop with "Western clarity ECL"
I have attached pictures of my developed film. Since this was just to test the efficacy of our antibodies, I cut the blot in half vertically. The left side I ran Alpha 2 and the right side of the blot I ran Alpha 4.
The band you see may not even be GABA alpha 4. It could be a an adundant protein in your lysate that cross reacts strongly with your antibody - this is also picked up by the alpha 2 antibody. If you have a plasmid to express your protein in cells, or a siRNA/shRNA to block its expression, you may be able to see how specific is your antibody, and if can pick these changes in protein expression. Another approach is to enrich your protein of interest, by getting a membrane fraction of the lysate for example, so as to incease the chances of your antibody finding your protein. It may be that your protein is too dilute to be recognised by the antibody, and/or you antibody is not good enough at detecting it.
If you think that your beta-mercaptoethanol may not be working properly, you could also try fresh DTT (at least 25 mM, and you could go up to 100 or 200 mM) to reduce potential disulfide bonds. High concentrations of Urea in your sample buffer (around 8 M) could also contribute to better denaturation of our protein and the break up any bonds contributing to dimerization or SDS-resistant complexes. However, it's not recomended to boil your sample after additing urea.
Hope one of these helps! Good luck!Following
- Mohit Kumar added an answer:20I am having issues with my exogenously expressed membrane protein smearing on SDS PAGE gel.Has anyone had issues with protein band smearing? I reduce the amount of sample I load on the SDS PAGE gel, but all that changes is the intensity of the smear. My protein standard runs normally. I incubate my protein on ice for 30 minutes in the presence of 100mM DTT, which has no affect on the smearing pattern. Any advice/suggestions would be greatly appreciated.
i am facing the same problem in upper side of my gel means in high molecular wt proteins but lower proteins running well but most of the protein remain in smear thats why faint bands after staining.Following
- Asok Mukhopadhyay added an answer:5Has anyone the protocol for using mouse serum / plasma on western blot?
I've got serum samples from WT and KO mice and would like to check cytokine on western blot, I tried to mix serum with sample buffer but having precipitate after boiling, anyone have a detail protocol on that?
Cytokines are present in pg/ng level in serum, it will be wasteful effort to identify them in WB. The better approach will be ELISA that will provide you quantitative data. Alternatively, if you so keen in WB, remove albumin using UF cut 20 KD- 30 KD, concentrate the filtrate 10 folds and then run WB (I will not do that). All the best.Following
- Yoav Lubelsky added an answer:7How do I do a western blot a large molecular weight protein?
Recently, I am working on DNAPKcs phosphorylation,which is 450 kDa, I am struggling with detection in my WB(Western Blot).
First, our model organism is frog, so the first problem is the specificity of antibody.
I tried to use 5%,6% SDS gel, and add SDS in my transfer buffer. However, it seems not be working. Usually, there is no signal or a strong background that I cannot see any band.
Does anyone have some suggestions?
You said you're not sure about the specificity of the AB so that's where I'll start. You can try doing a dot-blot or running a control lane with extract from the species the antibody was raised against.
Increasing transfer time is a good idea. I have also found that positioning the gel in the transfer apparatus so the top of the gel is at the bottom give better transfer of the large proteins.Following
- Julie Lynn Horton added an answer:7Western blotting question: Are there any major differences in blocking steps between using the odyssey and a peroxidase conjugated secondary ab?
I recently switched between the odyssey imager (at another location) now to using a standard peroxidase reaction to image my westerns. I notice with the peroxidase reaction I get a serious amount of background, more so than with the odyssey. I am using a standard 5% BSA or 5% milk solution for 1 hour at RT. Has anyone had this problem before? (I am specifically looking at GIRK2 expression)
Makes sense! Thanks for the explanation John- I knew there was some sort of good reason for this :)Following
- Maddalena Romanelli added an answer:6Why do I get smeared bands in my western blot?
Although you may think that it is a simple question, but I am having trouble with this result. As you may see on the pict, the bands are smeared, which I don't know what is the reason behind.
For SDS Page, I ran in 30 CC, 1 Hours and for transfer: 144 CC 1.5 Hours
I hope you can help me :)
There may be a problem in your sample (which lysis buffer do you use for lysis?) or there may be a problem in immunoblot. For example you can try immunoblot your membrane with a different concentration of Abs or maybe you have a problem with blocking. Do you use milk or BSA for blocking? How long?Following
- Jeffrey Lakritz added an answer:6Why are my Western Blot antibodies binding to some lanes and not others?
Hi Everyone! Thank you for your help in advance.
I am working on purifying a few different proteins from both Ecoli and mammalian cells and am having the hardest time actually detecting them via Western Blot. Initially, I used an anti-V5 antibody from Thermo and it detected every single protein possible, so much so that it highly resembled the coomassie stain. After a bit of trouble shooting I got a refund and moved onto an Anti His Antibody instead (constructs have both tags).
As shown in the coomassie stain below, I am very sure that at least the second protein (boxed in red), if not 3 out of the 4 proteins I transformed, was expressed in all 3 lanes for my Ecoli transformations (the soluble fraction, the pellet, and the elution). However, an anti-His Western Blot seems to only be detecting the pellet lanes. 12 ug of protein was loaded in 13 uL into every well on both the coomassie gel and the western blot gel so I am not sure why the discrepancy between the different lanes is present.
I then tested if the concentration of the elution might have been too high by performing a 5 fold dilution on two proteins: the elution lane of the protein that was boxed in red in the coomassie stain and another purified protein that also had a His Tag as a positive control. Within 4 lanes I diluted the proteins from 12 ug--> 0.096 ug. After developing for 900 seconds, the most I could see was a single band of my original elution at the original concentration (see picture entitled WB troubleshooting).
My questions are:
1. Why would the same exact dilution of this antibody see the same exact sample at the same exact dilution on the troubleshooting blot but not the original blot.
2. Why would the antibody then not detect the second protein on the WB troubleshooting blot?
3. Should I just toss the antibody as trash or am I possibly messing up a part of the procedure?
4. Where there is a will, is there really a way?
Please note that this is using Anti-His unconjugated clone #RM146 from Millipore Lot QVP15. 03209. Cat: 04-1664. 1:5,000 dilution in TBST with 4% milk (found to be the best concentration by another lab) as a primary antibody and ECL anti Rabbit IgG HRP #NA9340V from GE Healthcare, 1:20,000 in TBST, as a secondary. I incubated overnight at 4 C in blocking buffer and 1 hour at room temp for both antibodies with 3 rounds of 10 minute TBST washes in between. I developed the blot using the Thermo ECL High Sensitivity Kit.
Any and all suggestions are needed and appreciated!
I think using Dr. Pettersen suggestions of using shorter blocking step and then complete primary antibody binding at 4C overnight. Improves specificity of process.
I would also check pH of your buffer to ensure the antibody binds to His-tag? Check insert supplied with antibody or call Millipore technical service?
Finally, we have had similar problems when trying new antibodies. Since you appear to have moderate amounts of protein, could you try to immunoprecipitate proteins using antibodies bound to magnetic beads. Elute bound proteins off beads and run on gel?Following
- Sultan Asad added an answer:8The case of the disappearing proteins from an immunoblot.I can successfully probe for 1 protein but NOTHING else after (actin, gapdh or tubulin). I combined the primary antibodies of actin and my protein of interest and this seems to skirt around the problem. Though, I'm curious to see if anyone else has had this issue and if they nailed the culprit(s). FYI, I use laemmli lysis buffer, sonicate the cells, 10% sds-gel (made with new reagents), Ponceau staining looks fine (image attached) on nitrocellulose membrane, block in 3% milk and probe with primary antibody in BSA, and we have new ECL detection reagents. I've tried increasing the ratio of primary to BSA and nothing works. What gives?
I usually use two antibodies (one internal control HSP70 and other one specific antibody) but usually I use antibodies raised in different species. For example if I use control antibody from mouse I use specific antibody from other species like rabbit. Usually it work perfectly in nitrocellulose membrane.Following
About Western Blot
Western blot is a widely accepted analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein.