Technique / Proteomics and protein biochemistry / Western blot assay

Western blot problem

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	By AC on 14-Feb-2008 I am having trouble with my western blot. I use high concentrations of lysate and antibody. However, I am getting blank results. Can anyone tell me what may be the problem? Rating: n/a Reply

	By HongLien on 23-Sep-2007 I used Western blot to detect MMP-10 in cultured VSMC but did not receive any signal on film, Please advise me. Many thanks Rating: n/a Reply

	By Fontenele on 02-May-2007 Mahboubeh, do you have a very well protein extraction in your Lab. In my work some proteins require different extraction protocol (ie. different lysis buffer). Rating: Good Reply

	By Mahboubeh on 26-Mar-2007 I face with a problem in western blot.when I transfer proteins to nitrocellulose membrane it can not be detected protein with DAB or ECL solution,could you kindly help me?meanwhile my prestained molecular weight marker is transfered well. Rating: Good Reply

	By Hem Chandra Jha on 14-Jul-2005 Hi, I am facing one problem, I don't know about , How much quintity of protein load in SDS -PAGE when I transfer to PVDF membrain. Is this variable to organism to organism. Rating: n/a Reply

	By Timea on 16-Mar-2005 Hi, I would like to know, how can I quantify my western blot. I dont want to know the real amount of protein, only compare my samples. Can I do it by comparing the intensity of bands on the membrane, wich I have scaned before? Thank you very much Rating: Good Reply

By Sana on 07-Oct-2004 Everything in western blot is working fine EXCEPT my gel/samples. I dont see my front line going down as straight blue line, i see instead blue dots...and my lanes expand horizontally so at the end of western blot, I see connecting lines (impossible to differentiate between lanes..!!)why is that? is my gel technique messed up or is it my sample? or is it my calculation? maybe someone can tell me how they calculate out and prepare there samples for loading..( I use 5xsample buffer...loading buffer and protein sample x....) I extract my protein using RIPA(I make it myself)..so what i do is wash cells in PBS, scrap cells in PBS, centrifuge and then you resuspend your pellet in x volume of Ripa with freshly added inhibitors..Next once all plates are done and all microfuge tubes are on ice..I homogenize them using pasteur pipete and then add PMSF and sodium orthovandate..vortex and leave on ice for 30 min...centrifuge at max for 20 min and the supernatant is what I keep.... What I do is, I prepare my 12% separating gel a night before I am using. I am using 2.5ml pf 1.5 M Tris, pH 8.8 dH20=3.35ml 10% SDS 100 ul 30% Acrylamide 4ml when I am ready to use it, I add freshly prepared 10% APS 100ul and TEMED 7.5ul..Mix! Layer it with water or ethanol to get straight line and get rid of bubbles I let is polymerize for 30min+ and leave it in 4degrees O/N Next day: prepare stacking gel(4%)2.5ml of 0.5M Tris,pH 6.8, dH20 6.10ml, 10% SDS 100ul, 30% acrylamide 1.30ml when ready, pour off water or ethanol from separating gel...add TEMED 10ul and fresh APS 100ul to the stacking gel solution. Mix gently and pour in..insert comb and leave it for 30+min to polymerize. Please help! Regards, SJ Rating: Good Reply

	By azam on 28-Sep-2004 When I transfer into nitrocellulose paper Pre stain marker does not transfer properly then it is difficult to point out marker position and I have another problem during transfer voltage increase and electricity flow decrease.If any body have experience please suggest me. Rating: Good Reply

	By Lin Yihui on 01-Aug-2003 I found that the Histone 3(<15KD)was not easy to transfer to Nitrocellulose,help me! Rating: Good Reply

By zhong on 22-Apr-2003 Hi, I tried to detect EGFR (>170 kDa) on the cell membrane. I know there are EGFR on the membrane since I detected them by confocal microscopy. And the positive control (EGFR purchased from Sigma) can be detected in the western blot. I boiled the cultured cell pellet in SDS buffer for about 5 minutes, but can not detect them. Who can give me some suggestion about detect membrane receptor by western blot. Thanks. Rating: Good Reply

By zhong on 22-Apr-2003 Hi, I tried to detect EGFR (>170 kDa) on the cell membrane. I know there are EGFR on the membrane since I detected them by confocal microscopy. And the positive control (EGFR purchased from Sigma) can be detected in the western blot. I boiled the cultured cell pellet in SDS buffer for about 5 minutes, but can not detect them. Who can give me some suggestion about detect membrane receptor by western blot. Thanks. Rating: Good Reply

	By janak77 on 16-Apr-2003 hi i tried to seperate my 45 kd protein from kidney. but i didnt get expression. what are the main reasons behind that. i tried everything. my gel was running fine. it transfered well but in final step i didnt get anything?? help me out !!!! Rating: Good Reply

	By janak77 on 16-Apr-2003 hi i tried to seperate my 45 kd protein from kidney. but i didnt get expression. what are the main reasons behind that. i tried everything. my gel was running fine. it transfered well but in final step i didnt get anything?? help me out !!!! Rating: Good Reply

By congmiao on 18-Feb-2003 I have the same problem of the poor transfer onto the membrane. I use 7.5% SDS page gel. 190KD band could not be transfered onto the membrane when I stain the gel after transfer. I use . I trsfer buffer is tris, glycine, SDS and methanol buffer. Who could give me some idea. and by the way, if the protein charge influence the transfer ? If the protein supposed to be a negative charged protein, it should be like what... Most of the protein should be nagetive charged... Now I try to find solution ... thanks. Rating: Good Reply

By congmiao on 18-Feb-2003 I have the same problem of the poor transfer onto the membrane. I use 7.5% SDS page gel. 190KD band could not be transfered onto the membrane when I stain the gel after transfer. I use . I trsfer buffer is tris, glycine, SDS and methanol buffer. Who could give me some idea. and by the way, if the protein charge influence the transfer ? If the protein supposed to be a negative charged protein, it should be like what... Most of the protein should be nagetive charged... Now I try to find solution ... thanks. Rating: Good Reply

	By yingliwu Dr on 06-Dec-2002 i agree with SwingDr,i work on a protein of 210kd,and it can be transfered efficiently. Rating: Good Reply

	By nmatta on 23-Nov-2002 Hi, I´m having problems with the time of running, Im using acrilamida10%, running buffer with (Glycine, SDS, and tris), the total time its about 10 hours. Does anyone can help me Thanks Nubia Rating: Good Reply

	By hemaraina on 11-Jul-2002 hi I am also facing a similar problem of transferring a protein of high mol wt. I am working on 120 KD protein. I hope the previous comments will help. Apart form that I am having a fair bit of background with it.......... any suggestions Dr. hema raina Rating: Good Reply

By james on 08-Mar-2002 hi, I just wonder whether the protein will react in the same manner while it is in solution and in the gel. I quantified the protein by bradford and based on the result i loaded equal amount of protein in the wells of 14% acrylamide gel, but the gell pattern does not look like that i have loaded equal amount of protein. Do u have any comment for that? james Rating: Good Reply

	By V on 03-Oct-2001 I'm working with a 113 kd protein and I have had no problem. I use a 10% gel too. I add 2ml of 10% SDS to 2L of transfer buffer which is Tris based. Rating: Good Reply

	By Dr. renu walia on 06-Sep-2001 i too am facing a similar problem of transfering a big protein of 172 Kd in wet transfer buffer overnight at 4 deg at 100mA. i satined the gel after tranfer and foung my protein was still on the gel. my transfer buffer is Tris Base SDS and not glycine. Rating: Good Reply

By SwingDr on 22-Jun-2001 Having similar problems with transfer efficiency of a 200 kDa protien. Referencing "Molecular Cloning, A laboratory Manuel " (Sambrook, Fritsch, Maniatis) They suggest an effective range of separation of SDS-Polyacrylamide Gels as follows: 15% = 12-43 10% = 16-68 7.5% = 36-94 5% = 57-212 They also recommend the following Transfer Buffer: 39 mM Glycine 48 mM Tris Base 0.037% SDS (electrophoresis grade) 20% Methanol They have the transfer running with a current of 0.65 mA/sq. cm. of gel for a period of 1.5 to 2 hours. In several experiments I have seen better transfer of higher molecular weight proteins with the addition of SDS. Also we have had problems with poor grades of Tris base that give acidic pH once dissolved in water instead of the usual ~8.5. Another aspect that they repeatidly point to is the possiblity of a short by using nitrocelluse or stack paper that is larger than the gel itself. The make a big deal about having these exaclty the same size as the gel. Hope this is informative. Let me know if it works for you. Rating: Good Reply