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Discussion on troubleshooting on High background signal, Non-specific bands on the blot, Low sensitivity etc.
Posted by: biowww (IP Hidden, Regular member, 31)
Date: November 27, 2004 01:34PM
It includes troubleshooting on High background signal, Non-specific bands on the blot, Low sensitivity etc. (AgriSera)
Western blot troubleshooting [www.agrisera.se] <b>Table of Contents </b> Problems: High background signal Non-specific bands on the blot Low sensitivity Is a right band detected? Uneven results with lots of spots Note: Following are old discussion threads moved from the old server. | Printer-friendly page | Send this article to a friend | Comment List -------------------------------------------------------------------------------- Topic: Author: Time: spots instead of a band probing for Keratin 10 Jim J 26.11.2004 14:22 I have probed for keratin 10 on PVDF membrane, using 2Dgel lysate suspended in lamaelli buffer. I get multiple spots instead of a continuous band at the correct molecular weight. Does anyone know why, and what I should do to prevent it? Thanks, Jim Extra band at ¡À 120 kDa in sample buffer Harry Gosker 29.07.2004 07:37 We experience something funny. We run normal SDS page gels. On our blots, we see an extra band (roughly estimated)at 120 kDa. It also appears in lanes in which we just loaded sample buffer (mercapto-eth, glycerol, bromophenol blue and Tris). Sample buffer was freshly made several times. Band appears using different secondary (peroxidase labeled) antibodies. Omitting primary antibody still yields the extra band. Anybody knows what's going on? no signal for high molecular weight protein of 384kDa Neeraj Jain 10.07.2004 19:09 I am facing problems in getting signal for high molecular weight protein like BRCA2 which is of 384kDa.I would like to know how much percent stacking and resolving gel should be used and for how long semi dry transfer should be performed. I am using PVDF membrane (Millipore), santascruz antibodies 1:3000dilution for primary and 1:5000 dilution for secondary and ECL for detection. no bands after developing saritha tunuguntla 21.05.2004 08:33 Hi I have used a stained gel[with Commassie] for western blot wet transfer.I am confident that bands transferred on to the membrane since I can visibly see them as the gel was stained. However, after Ab & secondary Ab treatments with washings & ECL kit ,I see absoultely nothing on my X-ray film. I have tried 15s,30s,1m,5m,15m exposures. I used HRPconjugated Ab's. Both primary & secondary Ab dilutions have been tried in 5% non fat dried milk in TBS-T & also in 0.05% milk in TBS-T.Blocking agent is 5% non fat dried milk. Any inputs on this one???? Thank you Sari RE:no bands after developing 27.07.2004 18:18 > > Hi > > I have used a stained gel[with Commassie] for western blot > wet transfer.I am confident that bands transferred on to the > membrane since I can visibly see them as the gel was > stained. > > However, after Ab & secondary Ab treatments with washings & > ECL kit ,I see absoultely nothing on my X-ray film. > > I have tried 15s,30s,1m,5m,15m exposures. > I used HRPconjugated Ab's. > Both primary & secondary Ab dilutions have been tried in 5% > non fat dried milk in TBS-T & also in 0.05% milk in > TBS-T.Blocking agent is 5% non fat dried milk. > > Any inputs on this one???? > > Thank you > Sari > hi sari, i have had the same problem. maybe your AB are not specific enough. Also, see if your ECL kit is a little old. RE:no bands after developing Mette Petersen 09.08.2004 03:20 > > > > Hi > > > > I have used a stained gel[with Commassie] for western > blot > > wet transfer.I am confident that bands transferred on to > the > > membrane since I can visibly see them as the gel was > > stained. > > > > However, after Ab & secondary Ab treatments with washings > & > > ECL kit ,I see absoultely nothing on my X-ray film. > > > > I have tried 15s,30s,1m,5m,15m exposures. > > I used HRPconjugated Ab's. > > Both primary & secondary Ab dilutions have been tried in > 5% > > non fat dried milk in TBS-T & also in 0.05% milk in > > TBS-T.Blocking agent is 5% non fat dried milk. > > > > Any inputs on this one???? > > > > Thank you > > Sari > > > > hi sari, > i have had the same problem. maybe your AB are not specific > enough. Also, see if your ECL kit is a little old. > Hi sari Probably it is not a good idea to stain the gel with coomassie prior to blotting. It could block the binding of your antibody. I always stain my blot with ponceau after transfer, which is a much more gentle stain for proteins and it is reversible. Another possibility is to stain your gel with coomassie and then cut out the band of interest. Put it in 50% ethanol for 2 days in the freezer (this will shrink the gelslice) and transfer the gelslice to another polyacrylamide gel. During the electrophoresis the protein will migrate out of the gelslice and into your gel. After electrophoresis is complete, you can blot your gel and perform the western as usual. This has worked for me using NBT/BCIP development. You can even perform an 'in gel digest' using an enzyme that cuts 1-4 times within your protein and get fragments that will still be recognized by your antibody. In this way, you have an even stronger indication that your antibody is actually binding to your protein of interest. Good luck Mette Secondary Antibody and Serum 11.05.2004 06:19 You have said in the Article of Western blot troubleshooting that it seems not good for blocking membarne with the serum same as secondary antibody comes from. Could you explain why? Triton X-100 & Bradford reagent problem? joanne chang 03.05.2004 00:25 Hello, for Western bloting I prepaired the samples with lysis buffer. The lysis buffer contained 1% Triton X-100. My question is, may the Bradford Reagent (for determination of protein concentration) be influence by Triton X-100 ? thank you for your help Joanne RE:Triton X-100 & Bradford reagent problem? 25.11.2004 19:49 Hello, yes if u use 1% Tx-100 it would react with your bradford reagent.you will see a blue color with 1% tx-100 alone also as you would see with a protein sample. Instead, you can use 0.1%-0.5% Tx-100. Sudha RE:Triton X-100 & Bradford reagent problem? 25.11.2004 19:49 Hello, yes if u use 1% Tx-100 it would react with your bradford reagent.you will see a blue color with 1% tx-100 alone also as you would see with a protein sample. Instead, you can use 0.1%-0.5% Tx-100. Sudha RE:Triton X-100 & Bradford reagent problem? 25.11.2004 19:48 Hello, yes if u use 1% Tx-100 it would react with your bradford reagent.you will see a blue color with 1% tx-100 alone also as you would see with a protein sample. Instead, you can use 0.1%-0.5% Tx-100. Sudha concentrate protein 31.03.2004 06:53 my samples for western blotting are too diluted with lysis buffer so that the protein concentration of the samples is too low.Do somebody know how to concentrate the protein when the samples are ready mixed with sample buffer (loading buffer)? weird black dots Melina 22.03.2004 13:11 I've had the problem recently that instead of an empty (white) space between bands, I have a black dot. Does anyone know what can be the reason for that? Thank you! RE:weird black dots G Zhou 24.03.2004 18:52 > I've had the problem recently that instead of an empty > (white) space between bands, I have a black dot. Does anyone > know what can be the reason for that? > Thank you! > > R the dots everywhere on your membrane? if so, 1. pellet antibodie, use super only. 2. wash every thing before you transfer. Ge unspecific band 04.08.2002 16:09 i am able to see a band of exactly 14.5 Kd in my SDS PAGE. it appears some times and dose not appear some time with silverstaining. it never appears with coomasie. if i do a westernblotting of that i am able to detect it with the same antibodies as that against my protein which is 18.6Kd protein. i am not able to judge whether it is an artifact or is it a real band chopped off from my protein? what could be the possible reason of it being chopped off in such a way if true.and if so the case why am i not seeing it every time i run the same sample. 2002-07-19 22:45:53 reena (reena_rathod@rediffmail.com) RE:unspecific band 08.05.2004 01:51 > i am able to see a band of exactly 14.5 Kd in my SDS PAGE. > it appears some times and dose not appear some time with > silverstaining. it never appears with coomasie. if i do a > westernblotting of that i am able to detect it with the same > antibodies as that against my protein which is 18.6Kd > protein. i am not able to judge whether it is an artifact or > is it a real band chopped off from my protein? what could be > the possible reason of it being chopped off in such a way if > true.and if so the case why am i not seeing it every time i > run the same sample. > > 2002-07-19 22:45:53 > reena (reena_rathod@rediffmail.com) Great Work! Cialis RE:unspecific band G Zhou 24.03.2004 19:03 > i am able to see a band of exactly 14.5 Kd in my SDS PAGE. > it appears some times and dose not appear some time with > silverstaining. it never appears with coomasie. if i do a > westernblotting of that i am able to detect it with the same > antibodies as that against my protein which is 18.6Kd > protein. i am not able to judge whether it is an artifact or > is it a real band chopped off from my protein? what could be > the possible reason of it being chopped off in such a way if > true.and if so the case why am i not seeing it every time i > run the same sample. > > 2002-07-19 22:45:53 > reena (reena_rathod@rediffmail.com) Well, there are lots of reason for a protein be chopped off in that way. None of the staining methods is able to sure you everything. If U really want to see it, try a MALDI, which is a cheap way to do it. Ge Edited 4 times. Last edit at 01/18/05 06:45PM by biowww.
protocol of ECL reagent preparation
Posted by: S ghost (IP Hidden, Unregistered user, )
Date: December 6, 2004 03:37AM
Dear Sir/ madam,
I shall be happy if anybody will give me the protocol of ECL reagent. How do I prepare ECL reagent by using luminol,cumuric acid ,Hydrogen peroxide ,Tris.What will be the stock. thanking you
protocol of ECL reagent preparation
Posted by: S ghost (IP Hidden, Unregistered user, )
Date: December 6, 2004 03:39AM
Dear Sir/ madam,
I shall be happy if anybody will give me the protocol of ECL reagent. How do I prepare ECL reagent by using luminol,cumuric acid ,Hydrogen peroxide ,Tris.What will be the stock. thanking you
Re: Discussion on troubleshooting on High background signal, Non-specific bands on the blot, Low sensitivity etc.
Posted by: Teresa de Leon (IP Hidden, Unregistered user, )
Date: December 9, 2004 05:48AM
I am doing nothern blot for 18S and rubisco using the Ambion kit Ultrahyb and psoralen-biotin labelling kit for probe. I'm already getting distinct band however, the background in each lanes are also high. what can I do to reduce or remove those background?
Re: Discussion on troubleshooting on High background signal, Non-specific bands on the blot, Low sensitivity etc.
Posted by: ann poblenz (IP Hidden, Unregistered user, )
Date: December 15, 2004 03:17PM
Here is my recipe for home made ECL:
STOCKS: 1) Luminol (Sigma #A8511) Dissolve 0.44 grams in 10ml of DMSO (store at 4 degrees C) 2) Coumaric Acid (Sigma # C9008) Dissolve 0.15 grams in 10ml DMSO (store at 4 degrees C) 3) 1M Tris-HCl, pH 8.5 4) Hydrogen Peroxide, 30% Solution A: Mix 1ml 1M Tris-HCL pH 8.5, 9ml of ddH2O, 100ul Luminol stock and 44ul Coumaric Acid stick. Mix well. Solution B: Mix 1ml 1M Tris-CL pH 8.5, 9ml of ddH2O and 6.2ul 30% Hydrogen Peroxide stock. Mix well. These should be done just before using them. (I have had sucssess keeping Soln. - A and -B frozen separately in aliquotes, sized as above). Mix solution A and B and do the rest of the ECL reaction as normal (keep the blot in the reagent for around 2-3 minutes before exposure. Signal is stable for about 30 minutes and is about 5 times as bright as Amershams ECL kit.
Re: protocol of ECL reagent preparation
Posted by: biowww (IP Hidden, Regular member, 31)
Date: December 18, 2004 12:08AM
Follow this link to download manual for ECL detection.
[www4.amershambiosciences.com] Good luck. S ghosh Wrote: ------------------------------------------------------- > Dear Sir/ madam, > I shall be happy if anybody will give me the > protocol of ECL reagent. How do I prepare ECL > reagent by using luminol,cumuric acid ,Hydrogen > peroxide ,Tris.What will be the stock. > thanking you Edited 1 times. Last edit at 12/18/04 12:12AM by biowww.
Protein slot blot problem
Posted by: kcm139 (IP Hidden, New member, 1)
Date: January 12, 2005 05:05PM
We need help trouble-shooting a protein slot blot. We are having a problem with non-specific binding of the antibody to the spots containing no protein. We are using a apparatus that applies a vaccuum to pull the sample through the membrane and we are using nitrocellulose-ECL. I think the problem is that the spots get over-dried during the vaccuuming. The samples do not filter through evenly (samples with more protein take longer) so some sample spots may be getting too dry. Do you have any suggestions on how to eliminate this problem?
Thanks, Kelly
Re: Discussion on troubleshooting on High background signal, Non-specific bands on the blot, Low sensitivity etc.
Posted by: Alison Leung (IP Hidden, New member, 1)
Date: August 4, 2005 12:42PM
Hi I get a really high background for my dot blot. I think there is something wrong with the way I am doing the blocking. What am I doing wrong such that the blocking doesnt work and everything becomes all dark? Could it be that the membranes did not dry completely ie did not absorb all the samples because only the samples are not detected in my blot for some odd reason!
Please help me. Thank you and have a great day! Alison:)
Re: Discussion on troubleshooting on High background signal, Non-specific bands on the blot, Low sensitivity etc.
Posted by: Kareema (IP Hidden, Unregistered user, )
Date: August 10, 2005 09:41AM
Hello Everybody,
I dont have enough experience in Western Blot. So I am wndering if you can help me. The problem is the following: I want to use a mouse antibody to detect a mouse protein in my blot (I have no other choices). I was informed that I may have problem later when using secondary antibody which will reconize all kind of mouse proteins in my blot. Do you think I still can order this mouse antibody and use it, without having problems? thank you very much for your help. Kareema
Re: Discussion on troubleshooting on High background signal, Non-specific bands on the blot, Low sensitivity etc.
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 15, 2005 04:38PM
if you truly have no choice, order the secondary antibody. be sure though and check for non-specific secondary antibody binding. to do this block your blot, omit the primary antibody, incubate with secondary antibody, wash, and proceed with detection. this will let you know if your secondary antibody is detecting all of the mouse proteins on your blot. if so you may want to use another method than western blot to detect your protein, if possible.
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