Western blot problem

I hope you are using Acrylamide:bisacrylamide in the ratio of 29:1, and not only 30%acrylamide alone.
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Hi, I am having problems with my film..after my western is done. I am working with NOS in the hippocampus, and when I did my film for 25 minutes I did not get any bands, but when if i stripp the membrane, then I see a band (not as strong though). What are the reasoning's behind this...how come I don't get any results the first time, but I get a band when I stripp? Is it due to antibody or improper amount of antibody-human error?
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Make sure that you keep buffer level maintaned. Initially I also had the same problem.
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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?


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I used Western blot to detect MMP-10 in cultured VSMC but did not receive any signal on film, Please advise me.
Many thanks
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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).
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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.
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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.
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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
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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
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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.
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I found that the Histone 3(<15KD)was not easy to transfer to Nitrocellulose,help me!


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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.
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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.
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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 !!!!
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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 !!!!
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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.
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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.
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i agree with SwingDr,i work on a protein of 210kd,and it can be transfered efficiently.
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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


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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
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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
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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.
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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.
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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.


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